CN113728008B

CN113728008B - anti-CD 40 antibodies and uses thereof

Info

Publication number: CN113728008B
Application number: CN202080027681.4A
Authority: CN
Inventors: 张宏恺; 王媛
Original assignee: Nankai University
Current assignee: Nankai University
Priority date: 2019-04-10
Filing date: 2020-04-10
Publication date: 2024-04-09
Anticipated expiration: 2040-04-10
Also published as: EP3954705A1; SG11202111188VA; JP7535060B2; CA3136491A1; EP3954705A4; CN113728008A; WO2020207470A1; AU2020256828A1; TW202103733A; US20220324988A1; JP2022527406A

Abstract

Novel antibodies and antibody fragments that specifically bind CD40 are provided, as are compositions, medicaments, combinations, and kits containing the antibodies or antibody fragments. In addition, nucleic acids encoding the antibodies or antibody fragments thereof and host cells comprising the same, as well as related uses, are provided. In addition, therapeutic and diagnostic uses of these antibodies and antibody fragments are provided.

Description

anti-CD 40 antibodies and uses thereof

The present invention relates to novel antibodies and antibody fragments that specifically bind CD40 and compositions containing said antibodies or antibody fragments, as well as medicaments, combinations of products or kits. Furthermore, the invention relates to nucleic acids encoding said antibodies or antibody fragments thereof and host cells comprising the same, as well as related uses. Furthermore, the invention relates to therapeutic and diagnostic uses of these antibodies and antibody fragments.

Background

Complete activation of T cells requires two signals: antigen Presenting Cells (APCs) uptake, process tumor antigens, form MHC-antigen complexes, present to T cells and bind to TCRs on the surface of T cells, which are the first signal for T cell activation; the second or co-stimulatory signal is transmitted by CD28 interacting with B7-1 (CD 80)/B7-2 (CD 86), and co-stimulatory factors CD40, OX40, GITR, etc. interacting with their ligands (Smith-Garvin, J.E., G.A.Koretzky, and M.S. Jordan, T cell activation. Annu Rev Immunol, 2009.27:p.591-619). In the absence of co-stimulatory signals, T cells may undergo non-responsiveness (anergy) or programmed cell death (apoptosis) following antigen stimulation.

CD40 is a member of the TNF receptor (TNFR) superfamily, and is predominantly expressed on a variety of antigen presenting cells such as B cells, dendritic cells (DC cells), monocytes and macrophages (Grewal, I.S. and R.A. Flavell, CD40 and CD154 in cell-mediated immunity. Annu Rev Immunol, 1998.16:p.111-35). CD40 forms trimers on the cell surface and the corresponding ligand CD40L (i.e. CD 154) is predominantly expressed on the surface of activated T cells. The interaction of CD40 and CD40L is a costimulatory signal for T cell activation. Depending on the particular cell type, CD40 involvement leads to a particular gene expression pattern. Binding of CD40L to CD40 on T cells activates a variety of pathways, including NF-. Kappa.B (nuclear factor-. Kappa.B), MAPK (mitogen activated protein kinase) and STAT3 (signal transducer and transcriptional activator 3), among others (Rothe, M.et al, TRAF2-mediated activation of NF-kappa B by TNF receptor 2 and CD40.Science,1995.269 (5229): p.1424-7).

CD40 is expressed not only by normal immune cells, but also by many malignant cells. Specifically, CD40 is overexpressed in the following diseases: NHL, chronic Lymphocytic Leukemia (CLL), hairy Cell Leukemia (HCL), hodgkin's disease, multiple myeloma, bladder cancer, kidney cancer, ovarian cancer, cervical cancer, breast cancer, lung cancer, nasopharyngeal cancer, malignant melanoma, etc. (Hassan, s.b., et a1., anti-CD40-mediated cancer immunotherapy: an update of recent and ongoing clinical three.immunopharmacol, 2014.36 (2): p.96-104).

CD40 agonist antibodies can combat tumor cells by a variety of mechanisms: first, CD40 agonist antibodies mediate stronger anti-tumor effects by activating the immune system. In particular, CD40 agonist antibodies can activate DC cells to increase their antigen presenting capacity, as evidenced by increased expression of co-stimulatory molecules such as the B7 family (CD 80, CD 86), and promotion of cytokine secretion such as interleukin 12, which would result in a significant T cell response (Fong, l.and e.g. engleman, dendritic cells in cancer immunotherapy.annu Rev Immunol, 2000.18:p.245-73); CD40 agonist antibodies promote proliferation of resting B cells, immunoglobulin class switching, antibody secretion, and have an effect on the development of germinal centers and survival of memory B cells, all of which are essential for humoral immune responses (Beatty, G.L., Y.Li, and K.B. Long, cancer immunotherapy: activating innate and adaptive immunity through CD40 agonists.expert Rev Anticancer Ther,2017.17 (2): p.175-186). Second, after binding to CD40 expressed on the surface of tumor cells, CD40 agonist antibodies mediate antibody-dependent cellular cytotoxicity (ADCC), and tumor cells that highly express CD40 are cleared directly from killer cells (Vondegheide, R.H. and M.J. Glennie, agonitic CD40 antibodies and Cancer therapy. Clin Cancer Res,2013.19 (5): p.1035-43). Third, CD40 agonist antibodies, upon binding to CD40 expressed on the surface of tumor cells, directly inhibit tumor growth and promote apoptosis, e.g., CD40/CD40L signaling pathways block the Cell cycle of tumor cells, stop cells in the G2-M phase, CD40/CD40L interactions also promote elevation of Fas expression on the surface of tumor cells, inhibit growth and promote apoptosis of highly expressed CD40 tumor cells via Fas/FasL signaling pathways (Eliopoulos, A.G., et al, CD40 induces apoptosis in carcinoma cells through activation of cytotoxic ligands of the tumor necrosis factor superfamity. Mol Cell Biol,2000.20 (15): p.5503-15).

Agonist antibodies to CD40 can be classified into two classes according to their agonism, the first class of CD40 agonism being independent of Fc receptor cross-linking (e.g., CP-870893 and CDX-1140), although the former exhibit encouraging anticancer efficacy, the presence of dose-limiting toxicity can lead to the development of systemic immune dysfunction, venous thromboembolism and cytokine release syndrome (Cytokine Release Syndrome, CRS); another class requires Fc receptor cross-linking to have CD40 agonistic activity. Tumor tissue and surrounding draining lymph nodes have more tumor-associated inflammatory cells present and fcγr2b receptors have more aggregation around tumor cells. Thus, such "cross-linked antibody" agonists have a higher tissue selectivity, and the antibodies produce a pronounced agonism in the tumor microenvironment, while the ability to act at normal tissue sites of the body is kept low, thus improving the safety window of treatment.

Thus, although some CD40 antibodies, such as the CP870893 of the gabbro pharmaceutical, already exist in the art, there is still a need for new CD40 antibodies with properties comparable to or better than existing antibodies, in particular CD40 antibodies relying on Fc receptor cross-linking, in particular antibodies with better anti-cancer properties and higher safety.

Disclosure of Invention

The present invention thus provides a novel antibody that binds CD40, in particular human CD40 or rhesus CD40, and antigen binding fragments thereof.

In some embodiments, an anti-CD 40 antibody or antigen-binding fragment thereof of the invention comprises a heavy chain variable region (VH), wherein the VH comprises

(i) As set forth in SEQ ID NO: 13. 58, 60, 62 or 14, or three Complementarity Determining Regions (CDRs) HCDR1, HCDR2 and HCDR3 contained in a VH shown in fig. 58, 60, 62 or 14

(ii) Sequences which together comprise at least one and not more than 5, 4, 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) in said three CDR regions relative to the sequence of (i), or

(iii) At least one and no more than 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) are included on HCDR3 relative to the sequence of (i).

In some embodiments, an anti-CD 40 antibody or antigen-binding fragment thereof of the invention comprises a light chain variable region (VL), wherein the VL comprises:

(i) As set forth in SEQ ID NO: 15. three Complementarity Determining Regions (CDRs) LCDR1, LCDR2 and LCDR3 contained in VL shown at 64, 66 or 16; or (b)

(iii) At least one and no more than 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) are included on LCDR3 relative to the sequence of (i).

In some embodiments, an anti-CD 40 antibody or antigen-binding fragment thereof of the invention comprises a heavy chain variable region VH and a light chain variable region VL, wherein

(a) The VH comprises

(ii) A sequence comprising at least one and no more than 5, 4, 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) together in said three CDR regions relative to the sequence of (i);

(iii) At least one and no more than 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) are included on HCDR3 relative to the sequence of (i); and/or

(b) The VL comprises:

(ii) A sequence comprising at least one and no more than 5, 4, 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) together in said three CDR regions relative to the sequence of (i); or (b)

In some embodiments, an anti-CD 40 antibody or antigen-binding fragment thereof of the invention comprises a heavy chain variable region VH and/or a light chain variable region VL, wherein

(a) The VH comprises

(i) As set forth in SEQ ID NO: 13. 58, 60 or 62, or three Complementarity Determining Regions (CDRs) HCDR1, HCDR2 and HCDR3 contained in a VH shown in fig. 58, 60 or 62

(b) The VL comprises:

(i) As set forth in SEQ ID NO: 15. three Complementarity Determining Regions (CDRs) LCDR1, LCDR2 and LCDR3 contained in VL indicated at 64 or 66; or (b)

(a) The VH comprises

(i) As set forth in SEQ ID NO:14, three Complementarity Determining Regions (CDRs) HCDR1, HCDR2 and HCDR3 contained in a VH shown in fig. 14, or

(b) The VL comprises:

(i) As set forth in SEQ ID NO:16, three Complementarity Determining Regions (CDRs) LCDR1, LCDR2 and LCDR3 contained in VL; or (b)

In a preferred embodiment, VH comprises an amino acid sequence selected from SEQ ID NOs: 13. 58, 60, 62 or 14, or a combination thereof.

In a preferred embodiment, VL comprises a sequence selected from the group consisting of SEQ ID NOs: 15. 64, 66 or 16, or a combination thereof.

In a preferred embodiment, the anti-CD 40 antibodies or antigen-binding fragments thereof of the invention comprise

As set forth in SEQ ID NO: 13. 58, 60, 62 or 14, and 3 complementarity determining regions HCDR of the heavy chain variable region as set forth in SEQ ID NO: 15. 64, 66 or 16.

As set forth in SEQ ID NO: 13. 58, 60 or 62, and 3 complementarity determining regions HCDR of the heavy chain variable region as set forth in SEQ ID NO: 15. 64 or 66, and 3 complementarity determining regions LCDR of the light chain variable region.

As set forth in SEQ ID NO:14, and 3 complementarity determining regions HCDR of the heavy chain variable region as set forth in SEQ ID NO:16, and 3 complementarity determining regions LCDR of the light chain variable region shown in seq id no.

In a preferred embodiment, the anti-CD 40 antibodies or antigen-binding fragments thereof of the invention comprise:

(i) As set forth in SEQ ID NO:13, and 3 complementarity determining regions HCDR1, HCDR2 and HCDR3 of the heavy chain variable region as set forth in SEQ ID NO:15, 3 complementarity determining regions LCDR1, LCDR2 and LCDR3 of the light chain variable region shown in figure 15;

(ii) As set forth in SEQ ID NO:58, and 3 complementarity determining regions HCDR1, HCDR2 and HCDR3 of the heavy chain variable region as set forth in SEQ ID NO:64, 3 complementarity determining regions LCDR1, LCDR2 and LCDR3 of the light chain variable region;

(iii) As set forth in SEQ ID NO:60 or 62, and 3 complementarity determining regions HCDR1, HCDR2 and HCDR3 of the heavy chain variable region as set forth in SEQ ID NO:66, 3 complementarity determining regions LCDR1, LCDR2 and LCDR3 of the light chain variable region;

(iv) As set forth in SEQ ID NO:14, and 3 complementarity determining regions HCDR1, HCDR2 and HCDR3 of the heavy chain variable region as set forth in SEQ ID NO:16, 3 complementarity determining regions LCDR1, LCDR2 and LCDR3 of the light chain variable region shown in figure 16;

(v) The CDR combination of any one of (i) - (iv), wherein at least one and no more than 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) are comprised in said sequence compared to HCDR3 and/or LCDR 3.

In some embodiments, an anti-CD 40 antibody or antigen-binding fragment thereof of the invention comprises a heavy chain variable region (VH) and/or a light chain variable region (VL), wherein

(i) The VH comprises Complementarity Determining Regions (CDRs) HCDR1, HCDR2 and HCDR3, wherein HCDR1 comprises SEQ ID NO:1 or 2, or consists of said amino acid sequence, or HCDR1 comprises an amino acid sequence corresponding to SEQ ID NO:1 or 2, has one, two or three changes (preferably amino acid substitutions, preferably conservative substitutions) in comparison to the amino acid sequence of 1 or 2; HCDR2 comprises SEQ ID NO:3 or 4, or consists of said amino acid sequence, or HCDR2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 3 or 4, has one, two or three changes (preferably amino acid substitutions, preferably conservative substitutions) in comparison to the amino acid sequence of seq id no; HCDR3 comprises SEQ ID NO: 5. 51, 52, 55 or 6, or HCDR3 comprises or consists of an amino acid sequence corresponding to SEQ ID NO: 5. 51, 52, 55 or 6, an amino acid sequence having one, two or three changes (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence;

and/or

(ii) Wherein the VL comprises Complementarity Determining Regions (CDRs) LCDR1, LCDR2, and LCDR3, wherein LCDR1 comprises SEQ ID NO:7 or 8 or consists of said amino acid sequence, or LCDR1 comprises an amino acid sequence corresponding to SEQ ID NO:7 or 8, has one, two or three changes (preferably amino acid substitutions, preferably conservative substitutions) in comparison to the amino acid sequence of seq id no; LCDR2 comprises SEQ ID NO:9 or 10 or consists of said amino acid sequence, or LCDR2 comprises an amino acid sequence corresponding to SEQ ID NO:9 or 10, has one, two or three changes (preferably amino acid substitutions, preferably conservative substitutions) in the amino acid sequence compared to the amino acid sequence of the other; LCDR3 comprises an amino acid sequence selected from SEQ ID NOs: 11. 53, 54, 56 or 12, or LCDR3 comprises or consists of an amino acid sequence corresponding to SEQ ID NO: 11. 53, 54, 56 or 12, has one, two or three changes (preferably amino acid substitutions, preferably conservative substitutions) in the amino acid sequence.

In a preferred embodiment, the invention provides an anti-CD 40 antibody or antigen-binding fragment thereof comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein

(a) The VH comprises

(i) HCDR1, HCDR2 and HCDR3 comprising or consisting of the following sequences: SEQ ID NO: 1. SEQ ID NO:3 and SEQ ID NO:5, a step of; or alternatively

(ii) HCDR1, HCDR2 and HCDR3 comprising or consisting of the following sequences: SEQ ID NO: 1. SEQ ID NO:3 and SEQ ID NO:51; or alternatively

(iii) HCDR1, HCDR2 and HCDR3 comprising or consisting of the following sequences: SEQ ID NO: 1. SEQ ID NO:3 and SEQ ID NO:52; or alternatively

(iv) HCDR1, HCDR2 and HCDR3 comprising or consisting of the following sequences: SEQ ID NO: 2. SEQ ID NO:4 and SEQ ID NO:6, preparing a base material; or alternatively

(v) The HCDR combination of any one of (i) - (iv) wherein at least one and no more than 5, 4, 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) are comprised together in said three CDR regions; or alternatively

(vi) The HCDR combination of any one of (i) - (iv) wherein at said HCDR3 comprises at least one and no more than 3, 2 or 1 amino acid change (preferably an amino acid substitution, preferably a conservative substitution);

and/or

(b) The VL comprises

(i) LCDR1, LCDR2 and LCDR3 comprising or consisting of: SEQ ID NO: 7. SEQ ID NO:9 and SEQ ID NO:11; or alternatively

(ii) LCDR1, LCDR2 and LCDR3 comprising or consisting of: SEQ ID NO: 7. SEQ ID NO:9 and SEQ ID NO:53; or alternatively

(iii) LCDR1, LCDR2 and LCDR3 comprising or consisting of: SEQ ID NO: 7. SEQ ID NO:9 and SEQ ID NO:54; or alternatively

(iv) LCDR1, LCDR2 and LCDR3 comprising or consisting of: SEQ ID NO: 8. SEQ ID NO:10 and SEQ ID NO:12; or alternatively

(v) The LCDR combination of any one of (i) - (iv) wherein at least one and no more than 5, 4, 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) are included together in said three CDR regions; or alternatively

(vi) The LCDR combination of any of (i) - (iv) wherein at said HCDR3 comprises at least one and no more than 3, 2 or 1 amino acid change (preferably an amino acid substitution, preferably a conservative substitution).

(a) The VH comprises

(iv) The HCDR combination of any one of (i) - (iii) wherein at least one and no more than 5, 4, 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) are comprised together in said three CDR regions; or alternatively

(v) The HCDR combination of any one of (i) - (iii) wherein at said HCDR3 comprises at least one and no more than 3, 2 or 1 amino acid change (preferably an amino acid substitution, preferably a conservative substitution);

and/or

(b) The VL comprises

(iv) The LCDR combination of any of (i) - (iii) wherein at least one and no more than 5, 4, 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) are included together in said three CDR regions; or alternatively

(v) The LCDR combination of any of (i) - (iii) wherein at least one and no more than 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) are comprised in said LCDR 3.

(a) The VH comprises

(i) HCDR1, HCDR2 and HCDR3 comprising or consisting of the following sequences: SEQ ID NO: 2. SEQ ID NO:4 and SEQ ID NO:6, preparing a base material; or alternatively

(ii) The HCDR combination of (i), wherein at least one and no more than 5, 4, 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) are comprised together in said three CDR regions; or alternatively

(iii) A HCDR combination of (i), wherein the HCDR3 comprises at least one and no more than 3, 2 or 1 amino acid change (preferably an amino acid substitution, preferably a conservative substitution);

and/or

(b) The VL comprises

(i) LCDR1, LCDR2 and LCDR3 comprising or consisting of: SEQ ID NO: 8. SEQ ID NO:10 and SEQ ID NO:12; or alternatively

(ii) A LCDR combination of (i), wherein at least one and no more than 5, 4, 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) are included together in said three CDR regions; or alternatively

(iii) The LCDR combination of (i), wherein at said LCDR3 comprises at least one and no more than 3, 2 or 1 amino acid change (preferably an amino acid substitution, preferably a conservative substitution).

In a preferred embodiment, the invention provides an anti-CD 40 antibody or antigen-binding fragment thereof comprising

(i) HCDR1, HCDR2 and HCDR3 comprising or consisting of the following sequences: SEQ ID NO: 1. SEQ ID NO:3 and SEQ ID NO:5, and LCDR1, LCDR2, and LCDR3 comprising or consisting of: SEQ ID NO: 7. SEQ ID NO:9 and SEQ ID NO:11; or alternatively

(ii) HCDR1, HCDR2 and HCDR3 comprising or consisting of the following sequences: SEQ ID NO: 1. SEQ ID NO:3 and SEQ ID NO:51, and LCDR1, LCDR2 and LCDR3 comprising or consisting of: SEQ ID NO: 7. SEQ ID NO:9 and SEQ ID NO:53; or alternatively

(iii) HCDR1, HCDR2 and HCDR3 comprising or consisting of the following sequences: SEQ ID NO: 1. SEQ ID NO:3 and SEQ ID NO:52, and/or LCDR1, LCDR2 and LCDR3 comprising or consisting of: SEQ ID NO: 7. SEQ ID NO:9 and SEQ ID NO:54; or alternatively

(iv) HCDR1, HCDR2 and HCDR3 comprising or consisting of the following sequences: SEQ ID NO: 2. SEQ ID NO:4 and SEQ ID NO:6, and LCDR1, LCDR2, and LCDR3 comprising or consisting of: SEQ ID NO: 8. SEQ ID NO:10 and SEQ ID NO:12;

(v) The HCDR and LCDR combination of any one of (i) - (iv) wherein at least one and no more than 5, 4, 3, 2 or 1 amino acid change (preferably an amino acid substitution, preferably a conservative substitution) is co-contained on and/or on said three HCDR regions; or alternatively

(vi) The HCDR and LCDR combination of any one of (i) - (iv) wherein at least one and no more than 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) are comprised in said HCDR3 and/or LCDR 3.

(a) The VH comprises

(i) HCDR1, HCDR2 and HCDR3 comprising or consisting of the following sequences: SEQ ID NO: 1. SEQ ID NO:3 and SEQ ID NO:55; or (b)

(ii) A combination of HCDRs as in (i), wherein at least one and no more than 5, 4, 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) are included together on the three HCDR regions;

and/or

(b) The VL comprises

(iii) LCDR1, LCDR2 and LCDR3 comprising or consisting of: SEQ ID NO: 7. SEQ ID NO:9 and SEQ ID NO:56; or (b)

(iv) A combination of LCDRs as in (i), wherein at least one and no more than 5, 4, 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) are included together on said three LCDR regions.

In some embodiments, SEQ ID NO:55 as follows:

ext> Aext> -ext> Rext> -ext> Eext> -ext> Rext> -ext> Vext> -ext> Gext> -ext> Aext> -ext> Xext> 1ext> -ext> Pext> -ext> Text> -ext> Yext> -ext> Yext> -ext> Yext> -ext> Xext> 2ext> -ext> Xext> 3ext> -ext> DVext> whereinext> Xext> 1ext>,ext> Xext> 2ext> orext> Xext> 3ext> mayext> beext> anyext> aminoext> acidext>,ext> preferablyext> whereinext> Xext> 1ext> mayext> beext> Text>,ext> Next>,ext> Wext>,ext> Kext>,ext> Aext> orext> Yext>,ext> moreext> preferablyext> Text> orext> Next>;ext> Wherein x2 is preferably W or Y; wherein X3 may be W, Y, M, F, T, more preferably M, W or Y.

In some embodiments, SEQ ID NO:56 as follows:

M-X1-X2-L-X3-X4-P-Y-T, wherein X1, X2, X3 and X4 may be any amino acid, preferably wherein X1 may be Q, N or P, more preferably Q or N; x2 may be G, Q, F, S, Y or M; more preferably G or Q; x3 may be E, N, T, S or K; more preferably E or N; x4 may be T, Q, V, L, P or E; more preferably T, Q or V.

In a preferred embodiment, the invention provides an anti-CD 40 antibody or antigen-binding fragment thereof comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises Complementarity Determining Regions (CDRs) HCDR1, HCDR2 and HCDR3 and the VL Comprises (CDRs) LCDR1, LCDR2 and LCDR3, wherein the antibody or antigen-binding fragment thereof comprises a combination of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 as set forth in the following table (table a):

Table a: exemplary combinations of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 in the antibodies or antigen-binding fragments thereof of the invention

In some embodiments, an anti-CD 40 antibody or antigen-binding fragment thereof of the invention comprises a heavy chain variable region VH and/or a light chain variable region VL, wherein,

(a) Heavy chain variable region VH

(i) Comprising a sequence selected from the group consisting of SEQ ID NOs: 13. 58, 60, 62 or 14, an amino acid sequence having or consisting of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity; or alternatively

(ii) Comprising a sequence selected from the group consisting of SEQ ID NOs: 13. 58, 60, 62 or 14 or consists of the amino acid sequence of seq id no; or alternatively

(iii) Comprising a sequence selected from the group consisting of SEQ ID NOs: 13. 58, 60, 62 or 14, preferably said amino acid change does not occur in the CDR region, preferably said amino acid change occurs in the FR region, compared to an amino acid sequence having 1 or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, 1) amino acid changes (preferably amino acid substitutions, more preferably amino acid conservative substitutions);

and/or

(b) Light chain variable region VL

(i) Comprising a sequence selected from the group consisting of SEQ ID NOs: 15. 64, 66 or 16, an amino acid sequence having or consisting of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity;

(ii) Comprising a sequence selected from the group consisting of SEQ ID NOs: 15. 64, 66 or 16 or consists of the amino acid sequence of seq id no; or alternatively

(iii) Comprising a sequence selected from the group consisting of SEQ ID NOs: 15. 64, 66 or 16, preferably said amino acid change does not occur in the CDR region, preferably said amino acid change occurs in the FR region, compared to an amino acid sequence having 1 or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, 1) amino acid changes (preferably amino acid substitutions, more preferably amino acid conservative substitutions).

In some embodiments, the heavy chain variable region VH of an anti-CD 40 antibody or antigen-binding fragment thereof of the invention

(i) Comprising a sequence selected from the group consisting of SEQ ID NOs: 13. 58, 60 or 62, has or consists of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity; or alternatively

(ii) Comprising a sequence selected from the group consisting of SEQ ID NOs: 13. 58, 60 or 62 or consists of the amino acid sequence; or alternatively

(iii) Comprising a sequence selected from the group consisting of SEQ ID NOs: 13. 58, 60 or 62, preferably said amino acid change does not occur in the CDR regions, preferably said amino acid change occurs in the FR regions compared to an amino acid sequence having 1 or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, 1) amino acid changes (preferably amino acid substitutions, more preferably amino acid conservative substitutions).

In some embodiments, an anti-CD 40 antibody or antigen-binding fragment thereof of the invention comprises a light chain variable region VL

(i) Comprising a sequence selected from the group consisting of SEQ ID NOs: 15. 64 or 66, an amino acid sequence having or consisting of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity;

(ii) Comprising a sequence selected from the group consisting of SEQ ID NOs: 15. 64 or 66 or consists of the amino acid sequence of seq id no; or alternatively

(iii) Comprising a sequence selected from the group consisting of SEQ ID NOs: 15. 64 or 66, preferably said amino acid change does not occur in the CDR regions, preferably said amino acid change occurs in the FR regions compared to an amino acid sequence having 1 or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, 1) amino acid changes (preferably amino acid substitutions, more preferably amino acid conservative substitutions).

(i) Comprising a sequence selected from the group consisting of SEQ ID NOs: 14, or consists of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity; or alternatively

(ii) Comprising a sequence selected from the group consisting of SEQ ID NOs: 14 or consists of the amino acid sequence of seq id no; or alternatively

(iii) Comprising a sequence selected from the group consisting of SEQ ID NOs: 14, preferably said amino acid change does not occur in CDR regions, preferably said amino acid change occurs in FR regions, compared to an amino acid sequence having 1 or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, 1) amino acid changes (preferably amino acid substitutions, more preferably amino acid conservative substitutions).

(i) Comprising a sequence selected from the group consisting of SEQ ID NOs: 16, or consists of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity;

(ii) Comprising a sequence selected from the group consisting of SEQ ID NOs: 16 or consists of the amino acid sequence of seq id no; or alternatively

(iii) Comprising a sequence selected from the group consisting of SEQ ID NOs: 16, preferably said amino acid change does not occur in CDR regions, preferably said amino acid change occurs in FR regions, compared to an amino acid sequence having 1 or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, 1) amino acid changes (preferably amino acid substitutions, more preferably amino acid conservative substitutions).

(i) The heavy chain variable region VH comprises SEQ ID NO:13, and a light chain variable region VL comprising or consisting of the amino acid sequence of SEQ ID NO:15 or consists of the amino acid sequence of seq id no;

(ii) The heavy chain variable region VH comprises SEQ ID NO:58, and a light chain variable region VL comprising or consisting of the amino acid sequence of SEQ ID NO:64 or consists of the amino acid sequence of 64;

(iii) The heavy chain variable region VH comprises SEQ ID NO:60, and a light chain variable region VL comprising or consisting of the amino acid sequence of SEQ ID NO:66 or consists of the amino acid sequence of 66;

(iv) The heavy chain variable region VH comprises SEQ ID NO:62, and a light chain variable region VL comprising or consisting of the amino acid sequence of SEQ ID NO:66 or consists of the amino acid sequence of 66;

(v) The heavy chain variable region VH comprises SEQ ID NO:14, and a light chain variable region VL comprising or consisting of the amino acid sequence of SEQ ID NO:16 or consists of the amino acid sequence of 16.

In some embodiments, the amino acid sequence of the heavy chain variable region of an antibody of the invention has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of the heavy chain variable region of (i) - (v) described above, or an amino acid sequence thereof comprising 1 or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, 1) amino acid changes (preferably amino acid substitutions, more preferably amino acid conservative substitutions) compared to the amino acid sequence of the heavy chain variable region of (i) - (v) described above, preferably the amino acid changes do not occur in the CDR region, preferably the amino acid changes occur in the FR region.

In some embodiments, the amino acid sequence of the light chain variable region of an antibody of the invention has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of the light chain variable region of (i) - (v) described above, or an amino acid sequence thereof comprising 1 or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, 1) amino acid changes (preferably amino acid substitutions, more preferably amino acid conservative substitutions) compared to the amino acid sequence of the light chain variable region of (i) - (v) described above, preferably the amino acid changes do not occur in the CDR region, preferably the amino acid changes occur in the FR region.

In a preferred embodiment, the invention provides an anti-CD 40 antibody or antigen-binding fragment thereof comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the antibody or antigen-binding fragment thereof comprises a combination of a heavy chain variable region VH and a light chain variable region VL as set forth in the following table (table B):

table B: exemplary combinations of heavy chain variable region VH and light chain variable region VL in an antibody or antigen-binding fragment thereof of the invention

In some embodiments, an anti-CD 40 antibody or antigen-binding fragment thereof of the invention comprises a heavy chain and/or a light chain, wherein

(a) Heavy chain

(i) Comprising a sequence selected from the group consisting of SEQ ID NOs: 17. 18, 19, 67, 69, 70 or 20, has or consists of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity;

(ii) Comprising a sequence selected from the group consisting of SEQ ID NOs: 17. 18, 19, 67, 69, 70 or 20 or consists of the amino acid sequence; or alternatively

(iii) Comprising a sequence selected from the group consisting of SEQ ID NOs: 17. 18, 19, 67, 69, 70 or 20, preferably, the amino acid change does not occur in the CDR region of the heavy chain, more preferably, the amino acid change does not occur in the heavy chain variable region, most preferably, the amino acid change occurs in the heavy chain constant region, compared to an amino acid sequence having 1 or more (preferably, no more than 20 or 10, more preferably, no more than 5, 4, 3, 2, 1) amino acid changes (preferably amino acid substitutions, more preferably amino acid conservative substitutions);

and/or

(b) Light chain

(i) Comprising a sequence selected from the group consisting of SEQ ID NOs: 21. 68, 71 or 22, an amino acid sequence having or consisting of at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity;

(ii) Comprising a sequence selected from the group consisting of SEQ ID NOs: 21. 68, 71 or 22 or consists of the amino acid sequence of seq id no; or alternatively

(iii) Comprising a sequence selected from the group consisting of SEQ ID NOs: 21. 68, 71 or 22, preferably, said amino acid change does not occur in the CDR region of the light chain, more preferably, said amino acid change does not occur in the light chain variable region, most preferably, said heavy chain amino acid change occurs in the light chain constant region, compared to an amino acid sequence having 1 or more (preferably, no more than 20 or 10, more preferably, no more than 5, 4, 3, 2, 1) amino acid changes (preferably amino acid substitutions, more preferably amino acid conservative substitutions).

In some embodiments, an anti-CD 40 antibody or antigen-binding fragment thereof of the invention comprises a heavy chain

(i) Comprising a sequence selected from the group consisting of SEQ ID NOs: 17. 18, 19, 67, 69 or 70, an amino acid sequence having or consisting of at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity;

(ii) Comprising a sequence selected from the group consisting of SEQ ID NOs: 17. 18, 19, 67, 69 or 70 or consists of the amino acid sequence; or alternatively

(iii) Comprising a sequence selected from the group consisting of SEQ ID NOs: 17. 18, 19, 67, 69 or 70, preferably, said amino acid change does not occur in the CDR region of the heavy chain, more preferably, said amino acid change does not occur in the heavy chain variable region, most preferably, said heavy chain amino acid change occurs in the heavy chain constant region, compared to an amino acid sequence having 1 or more (preferably, no more than 20 or 10, more preferably, no more than 5, 4, 3, 2, 1) amino acid changes (preferably amino acid substitutions, more preferably amino acid conservative substitutions).

In some embodiments, an anti-CD 40 antibody or antigen-binding fragment thereof of the invention comprises a light chain

(i) Comprising a sequence selected from the group consisting of SEQ ID NOs: 21. 68 or 71, an amino acid sequence having or consisting of at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity;

(ii) Comprising a sequence selected from the group consisting of SEQ ID NOs: 21. 68 or 71 or consists of the amino acid sequence of seq id no; or alternatively

(iii) Comprising a sequence selected from the group consisting of SEQ ID NOs: 21. 68 or 71, preferably, the amino acid change does not occur in the CDR region of the light chain, more preferably, the amino acid change does not occur in the light chain variable region, most preferably, the heavy chain amino acid change occurs in the light chain constant region, compared to an amino acid sequence having 1 or more (preferably, no more than 20 or 10, more preferably, no more than 5, 4, 3, 2, 1) amino acid changes (preferably amino acid substitutions, more preferably amino acid conservative substitutions).

(i) Comprising a sequence selected from the group consisting of SEQ ID NOs: 20, or consists of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity;

(ii) Comprising a sequence selected from the group consisting of SEQ ID NOs: 20 or consists of the amino acid sequence of 20; or alternatively

(iii) Comprising a sequence selected from the group consisting of SEQ ID NOs: 20, preferably, the amino acid change does not occur in the CDR regions of the heavy chain, more preferably, the amino acid change does not occur in the heavy chain variable region, most preferably, the heavy chain amino acid change occurs in the heavy chain constant region, compared to an amino acid sequence having 1 or more (preferably, no more than 20 or 10, more preferably, no more than 5, 4, 3, 2, 1) amino acid changes (preferably amino acid substitutions, more preferably amino acid conservative substitutions).

(i) Comprising a sequence selected from the group consisting of SEQ ID NOs: 22, or a sequence having or consisting of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical;

(ii) Comprising a sequence selected from the group consisting of SEQ ID NOs: 22 or consists of the amino acid sequence of seq id no; or alternatively

(iii) Comprising a sequence selected from the group consisting of SEQ ID NOs: 22, preferably, the amino acid change does not occur in the CDR regions of the light chain, more preferably, the amino acid change does not occur in the light chain variable region, most preferably, the heavy chain amino acid change occurs in the light chain constant region, compared to an amino acid sequence having 1 or more (preferably, no more than 20 or 10, more preferably, no more than 5, 4, 3, 2, 1) amino acid changes (preferably amino acid substitutions, more preferably amino acid conservative substitutions).

In some embodiments, an anti-CD 40 antibody or antigen-binding fragment thereof of the invention comprises a heavy chain and/or a light chain, wherein,

(i) The heavy chain comprises SEQ ID NO: 17. 18, 19, 67, 69 or 70, and the light chain comprises or consists of the amino acid sequence of SEQ ID NO: 21. 68 or 71 or a variant thereof,

(ii) The heavy chain comprises SEQ ID NO: 17. 18 or 19, and the light chain comprises or consists of the amino acid sequence of SEQ ID NO:21 or consists of the amino acid sequence of seq id no;

(iii) The heavy chain comprises SEQ ID NO:67, and the light chain comprises or consists of the amino acid sequence of SEQ ID NO:68 or consists of the amino acid sequence of seq id no;

(iv) The heavy chain comprises SEQ ID NO:69 or 70, and the light chain comprises or consists of the amino acid sequence of SEQ ID NO:71 or consists of the amino acid sequence of seq id no;

(v) The heavy chain comprises SEQ ID NO:20, and the light chain comprises or consists of the amino acid sequence of SEQ ID NO:22 or consists of the amino acid sequence of seq id no.

In some embodiments, the amino acid sequence of the heavy chain of an antibody of the invention has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of the heavy chain in (i) - (v) above, or an amino acid sequence thereof comprising 1 or more (preferably no more than 20 or 10, more preferably no more than 5, 4, 3, 2, 1) amino acid changes (preferably amino acid substitutions, more preferably amino acid conservative substitutions) compared to the amino acid sequence of the heavy chain in (i) - (v) above, preferably the amino acid changes do not occur in CDR regions of the heavy chain, more preferably the amino acid changes do not occur in heavy chain variable regions, most preferably the amino acid changes occur in constant regions of the heavy chain.

In some embodiments, the amino acid sequence of the light chain of an antibody of the invention has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of the heavy chain in (i) - (v) above, or an amino acid sequence thereof comprising 1 or more (preferably no more than 20 or 10, more preferably no more than 5, 4, 3, 2, 1) amino acid changes (preferably amino acid substitutions, more preferably amino acid conservative substitutions) compared to the amino acid sequence of the light chain in (i) - (v) above, preferably the amino acid changes do not occur in CDR regions of the light chain, more preferably the amino acid changes do not occur in light chain variable regions, most preferably the amino acid changes occur in constant regions of the light chain.

In a preferred embodiment, the invention provides an anti-CD 40 antibody or antigen-binding fragment thereof comprising a heavy chain and a light chain, wherein the antibody or antigen-binding fragment thereof comprises a combination of heavy and light chains as set forth in the following table (table C):

table C: exemplary combinations of heavy and light chains in an antibody or antigen-binding fragment thereof of the invention

In some embodiments, the heavy and/or light chain of an anti-CD 40 antibody or fragment thereof of the invention further comprises a signal peptide sequence, e.g., comprising the amino acid sequence of SEQ ID NO:43 or consists of the amino acid sequence shown in 43.

In one embodiment of the invention, the amino acid changes described herein include substitutions, insertions or deletions of amino acids. Preferably, the amino acids described herein are changed to amino acid substitutions, preferably conservative substitutions.

In some embodiments, the alteration occurs in CDR regions (particularly CDR3 regions) of the heavy and/or light chain of an antibody. In some embodiments, there may be 1, 2, or 3 changes in a CDR region, e.g., CDR3 region.

In a preferred embodiment, the amino acid changes described in the present invention occur in regions outside the CDRs (e.g., in the FR). In certain embodiments, the alteration occurs in an FR region of the antibody, e.g., an FR region of the antibody heavy and/or light chain variable region, e.g., an FR1, FR2 or F4 region. In some embodiments, the alteration occurs in the FR2 region. In some embodiments, there may be 1, 2, or 3 changes to the FR region.

More preferably, the amino acid changes described herein occur in regions outside the heavy chain variable region and/or outside the light chain variable region, for example in the constant region of the heavy chain and/or light chain.

In some embodiments, the substitutions are conservative substitutions. Conservative substitutions refer to the substitution of one amino acid with another within the same class, e.g., the substitution of one acidic amino acid with another acidic amino acid, the substitution of one basic amino acid with another basic amino acid, or the substitution of one neutral amino acid with another neutral amino acid. Exemplary permutations are shown in table D below:

Table D

Original residue	Exemplary substitutions	Preferred conservative amino acid substitutions
			Ala(A)	Val、Leu、Ile	Val
Arg(R)	Lys、Gln、Asn	Lys
			Asn(N)	Gln、His、Asp、Lys、Arg	Gln
Asp(D)	Glu、Asn	Glu
			Cys(C)	Ser、Ala	Ser
Gln(Q)	Asn、Glu	Asn
			Glu(E)	Asp、Gln	Asp
Gly(G)	Ala	Ala
			His(H)	Asn、Gln、Lys、Arg	Arg
Ile(I)	Leu, val, met, ala, phe norleucine	Leu
			Leu(L)	Norleucine Ile, val, met, ala, phhe	Ile
Lys(K)	Arg、Gln、Asn	Arg
			Met(M)	Leu、Phe、Ile	Leu
Phe(F)	Trp、Leu、Val、Ile、Ala、Tyr	Tyr
			Pro(P)	Ala	Ala
Ser(S)	Thr	Thr
			Thr(T)	Val、Ser	Ser
Trp(W)	Tyr、Phe	Tyr
			Tyr(Y)	Trp、Phe、Thr、Ser	Phe
Val(V)	Ile, leu, met, phe, ala norleucine	Leu

In certain embodiments, the substitution occurs in the CDR regions of the antibody. Typically, the resulting variants have modifications (e.g., improvements) in certain biological properties (e.g., increased affinity) relative to the parent antibody and/or will have certain biological properties of the parent antibody that are substantially preserved. Exemplary substitution variants are affinity matured antibodies. In some embodiments, the substitution occurs in CDR3 regions of the heavy and/or light chain of the antibody. In some embodiments, there may be 1, 2, or 3 substitutions in the CDR3 region.

In certain embodiments, the substitution occurs in the FR region of the antibody, e.g., the FR region of the antibody heavy and/or light chain variable region, e.g., the FR1, FR2 or F4 region. In some embodiments, the substitution occurs in the FR2 region. In some embodiments, 1, 2, or 3 substitutions may be present in the FR region.

In certain embodiments, the invention provides antibodies comprising a variable region sequence disclosed herein or a variable region sequence having CDRs disclosed herein, and constant domains having a modified Fc region having an increased affinity for fcγriib compared to its affinity for other Fc receptors (i.e., activated receptors). Expected to have enhanced fcyriib properties Such agonistic anti-CD 40 antibodies that are specific exhibit excellent efficacy in cancer treatment and chronic infection (Li and Ravetch (2011) Science 333:1030; white et al (2011) J.Immunol.187:1754). Without being bound by theory, such fcyriib-specific agonist anti-CD 40 antibodies may promote cytotoxicity of CD8 by increasing ⁺ Proliferation of T cells and maturation of activated dendritic cells, resulting in an enhanced anti-tumor response to exhibit enhanced helper effects. Without being bound by theory, fcR-mediated enhancement of signal by the agonist CD40 antibodies due to the crosslinking of the present invention may be a major contributor to therapeutic efficacy. Crosslinking of FcR-conjugated CD40 agonist antibodies by the Fc portion of the antibody can increase signal intensity and thereby enhance cell activation.

Mutations in Fc sequences capable of producing enhanced affinity for fcyriib are known in the art, for example described in Yu et al (2013) j.am.chem.soc.135:9723 and WO 2014/184545, chu et al (2008) mol. Immunol.45:3926, and Mimoto et al (2013) Protein Engineering Design & Selection 26:589. the nomenclature for the location (numbering) of mutations in the Fc region is according to the EU index, as in Kabat et al (1981) Sequences of Proteins of Immunological Interest, 5 th edition Public Health Service, national Institutes of Health, bethesda, md.), which facilitates comparison of Fc sequences at equivalent positions in antibodies with different variable domain lengths.

Exemplary mutations in the Fc sequence include, for example, E233D, G237D, H268D, P271G, A, R, S267E, and/or L328F. In a preferred embodiment, the antibodies of the invention comprise a human IgG1 constant domain that enhances fcyriib-specific mutations, including E233D, G237D, H268D, P271G and a330R, or S267E and L328F. Exemplary sequences of antibody heavy chains comprising mutated human IgG1 constant domains that enhance fcyriib specificity are described, for example, in SEQ ID NO:18 or 19.

In certain embodiments, the antibodies provided herein can be further modified to contain other non-protein moieties known and readily available in the art. Moieties suitable for antibody derivatization include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol/propylene glycol copolymers, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1, 3-dioxane, poly-1, 3, 6-trioxane, ethylene/maleic anhydride copolymers, polyaminoacids (homo-or random copolymers), and dextran or poly (n-vinylpyrrolidone) polyethylene glycol, propylene glycol homopolymers, polypropylene oxide/ethylene oxide copolymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof.

In certain embodiments, the antibodies provided herein are altered to increase or decrease the degree to which the antibodies are glycosylated. The addition or deletion of glycosylation sites to an antibody can be conveniently accomplished by altering the amino acid sequence so as to create or remove one or more glycosylation sites. When an antibody contains an Fc region, the saccharide attached thereto may be changed. In some applications, modifications that remove unwanted glycosylation sites may be useful, for example, to remove fucose moieties to enhance antibody-dependent cellular cytotoxicity (ADCC) function (see Shield et al (2002) JBC 277:26733). In other applications, galactosylation modifications may be performed to modify Complement Dependent Cytotoxicity (CDC).

In certain embodiments, it may be desirable to produce cysteine engineered antibodies, such as "thioMAbs," in which one or more residues of the antibody are replaced with cysteine residues. Cysteine engineered antibodies may be generated as described, for example, in U.S. patent No. 7,521,541.

In some embodiments, an anti-CD 40 antibody or antigen-binding fragment thereof of the invention exhibits the same or similar binding affinity and/or specificity for CD40 as an antibody of the invention; and/or inhibit (e.g., competitively inhibit) binding of an antibody of the invention to CD40 and/or binding to the same or overlapping epitope as the antibody of the invention; and/or compete with the antibodies of the invention for binding to CD40; and/or has one or more biological properties of the antibodies of the invention.

In some embodiments, the anti-CD 40 antibodies of the invention are antibodies in the IgG1 form or in the IgG2 form or in the IgG3 form or in the IgG4 form.

In some embodiments, the anti-CD 40 antibody is a monoclonal antibody.

In some embodiments, the anti-CD 40 antibody is humanized. Different methods for humanizing antibodies are known to the skilled artisan, as reviewed by Almagro & Franson, the contents of which are incorporated herein by reference in their entirety (Almagro JC and Franson J (2008) Frontiers inBioscience 13:1619-1633).

In some embodiments, the anti-CD 40 antibody is a human antibody. Various techniques known in the art may be used to prepare human antibodies. Human antibodies are generally described in van Dijk and van de Winkel, curr. Opin. Pharmacol 5:368-74 (2001) and Lonberg, curr. Opin. Immunol 20:450-459 (2008).

In some embodiments, the anti-CD 40 antibody is a chimeric antibody.

In one embodiment, the anti-CD 40 antibodies of the invention also encompass antibody fragments thereof, preferably antibody fragments selected from the group consisting of: fab, fab '-SH, fv, single chain antibody (e.g.scFv) or (Fab') ₂ A single domain antibody, diabody (dAb), or a linear antibody.

In certain embodiments, the anti-CD 40 antibody molecule is in the form of a bispecific or multispecific antibody molecule. In one embodiment, the bispecific antibody molecule has a first binding specificity for CD40 and a second binding specificity for PD-1 or PD-L2 or OX40 or 4-1BB or GITR, or the like. In one embodiment, the bispecific antibody molecule binds to CD40 and TNF or IL-17. The multispecific antibody molecule may have any combination of binding specificities for the foregoing molecules.

In some embodiments, the antibodies of the invention have one or more of the following properties:

(1) Selectively binds CD40, which has a higher binding capacity than other TNFR family proteins, or which does not bind other TNFR family proteins; in some embodiments, the binding is detected using ELISA.

(2) Blocking the binding of CD40 to CD40 ligand (CD 40L).

(3) Activating CD40 expressing cells, e.g. activating the NFkappa-B signaling pathway, in crosslinked or constitutive form, preferably in crosslinked form; in some embodiments, detection is performed using flow cytometry; in some embodiments, the reporter cells used are cells expressing NF- κB-GFP and hCD 40;

(4) Binds to CD40, e.g., human CD40, with an equilibrium dissociation constant of less than or equal to about 5X 10 ^-7 M、4.5×10 ^-7 M、4.4×10 ^-7 M、4.3×10 ^-7 M、4.2×10 ^-7 M、4.1×10 ^-7 M、4×10 ^-7 M、3.9×10 ^-7 M、3.8×10 ^-7 M、3.7×10 ^-7 M、3.6×10 ^-7 M、3.5×10 ^-7 M, or 3.4X10 ^-7 M; in some embodiments, the measurement is a surface plasmon resonance technique.

(5) Specifically binds to CD40 on cells expressing CD40 (e.g., human CD40 or rhesus CD 40); in some embodiments, the EC50 of binding to CD40 (e.g., human CD40 and/or rhesus CD 40) is less than or equal to about 50nM, 40nM, 30nM, 20nM, 15nM, 14nM, 13nM, 12nM, 10nM, 9nM, 8nM. In some embodiments, the combination is detected using flow cytometry; in some embodiments, the CD40 expressing cell is a 293 cell, e.g., a 293FT cell.

(6) Induce apoptosis of tumor cells. In some embodiments, the EC50 is less than or equal to about 4nM, 3.5nM, 3nM, 2.9nM, 2.8nM, 2.7nM, or 2.6nM; in some embodiments, the measurement is performed using flow cytometry. In some embodiments, the tumor cell is a Raji cell or a Ramos cell.

(7) Has agonist activity, such as significantly activating (e.g., human) B cells or T cells or dendritic cells.

(8) Promote proliferation of (e.g., human) B cells or T cells.

(9) Enhancing the immune response.

(10) Inhibit tumor growth, preferably while maintaining the individual's body weight.

In some embodiments, the CD40 antibody has one or more of the properties described above when crosslinked.

In some embodiments, the invention also encompasses antibodies conjugated to other substances ("immunoconjugates").

In some embodiments, the other substance is, for example, a therapeutic agent or marker, such as a cytotoxic or immunosuppressive agent or a chemotherapeutic agent. Cytotoxic agents include any agent that is detrimental to cells. Examples of cytotoxic agents (e.g. chemotherapeutic agents) or other substances suitable for forming immunoconjugates are known in the art, see e.g. WO2017/004006 or WO2017/059243, etc.

In some embodiments, the tag is, for example, a tag sequence, such as a peptide. In a preferred embodiment, the tagged amino acid sequence is a hexahistidine peptide, such as the tag provided in the pQE vector (QIAGEN, inc.,9259 Eton Avenue,Chatsworth,CA,91311) and the like, many of which are commercially available. Such as Gentz et al, 1989, proc.Natl. Acad. Sci. USA86:821-824, for example, hexahistidine provides for convenient purification of fusion proteins. Other peptide tags for purification include, but are not limited to, hemagglutinin ("HA") tags, which correspond to epitopes derived from influenza hemagglutinin protein (Wilson et al, 1984, cell 37:767) and "flag" tags.

In other embodiments, the label may be a diagnostic or detectable agent. The resulting antibody conjugates can be used as part of a clinical test method (e.g., to determine the efficacy of a particular therapy) for monitoring or predicting the onset, formation, progression and/or severity of a disease or disorder. Detectable or diagnostic agents include, but are not limited to, various enzymes such as horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase; prosthetic groups such as streptavidin/biotin and avidin/biotin; fluorescent substances such as, but not limited to, umbelliferone, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; luminescent substances such as, but not limited to, luminol; bioluminescent materials such as, but not limited to, luciferase, luciferin and aequorin; radioactive substances, such as but not limited to iodine @, and ¹³¹ I、 ¹²⁵ I、 ¹²³ i and ¹²¹ i) The carbon is ¹⁴ C) Sulfur ³⁵ S, tritium ³ H) The indium is ¹¹⁵ In、 ¹¹³ In、 ¹¹² In and ¹¹¹ in, technetium ] ⁹⁹ Tc), thallium ²⁰¹ Ti, ga ] ⁶⁸ Ga、 ⁶⁷ Ga and Pd% ¹⁰³ Pd and molybdenum% ⁹⁹ Mo and xenon ¹³³ Xe and F ¹⁸ F)、 ¹⁵³ 5m、 ¹⁷⁷ Lu、 ¹⁵⁹ Gd、 ¹⁴⁹ Pm、 ¹⁴⁰ La、 ¹⁷⁵ Yb、 ¹⁶⁶ Ho、 ⁹⁰ Y、47Sc、 ¹⁸⁶ Re、 ¹⁸⁸ Re、 ¹⁴² Pr、 ¹⁰⁵ Rh、 ⁹⁷ Ru、 ⁶⁸ Ge、 ⁵⁷ Co、 ⁶⁵ Zn、 ⁸⁵ Sr、 ³² P、 ¹⁵³ Gd、 ¹⁶⁹ Yb、 ⁵¹ Cr、 ⁵⁴ Mn、 ⁷⁵ Se、 ¹¹³ Sn and Sn ¹¹⁷ Tin; and positron emitting metals and nonradioactive paramagnetic metal ions for use in various positron emission imaging procedures.

In some embodiments, the therapeutic agent comprises a chemotherapeutic agent, cytokine, cytotoxic agent, other antibody, small molecule drug, or immunomodulatory agent.

In addition, the antibody molecules of the invention may be conjugated to a therapeutic moiety (therapeutic agent) such as a radiometal ion, such as an alpha-emitter such as ²¹³ Bi or is useful for the generation of radioactive metal ions (including but not limited to ¹³¹ In、 ¹³¹ LU、 ¹³¹ Y、 ¹³¹ Ho、 ¹³¹ Sm) conjugated to a polypeptide. In certain embodiments, the macrocyclic chelator is 1,4,7, 10-tetraazacyclododecane-N, N ', N ", N'" -tetraacetic acid (DOTA), which may be attached to an antibody through a linker molecule. Such linker molecules are well known in the art and are described in Denardo et al, 1998,Clin Cancer Res.4 (10): 2483-90, each of which is incorporated by reference in its entirety.

Techniques for conjugation of therapeutic moieties to antibodies are well known, see, e.g., arnon et al, "Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy", introduced Monoclonal Antibodies And Cancer Therapy, reisfeld et al (eds.), pages 243-256 (Alan R.Lists, inc. 1985).

In some embodiments, the invention provides nucleic acids encoding any of the antibodies or fragments thereof described herein or any of the strands thereof. In one embodiment, a vector comprising the nucleic acid is provided. In one embodiment, the vector is an expression vector, such as a pFuse vector. In one embodiment, a host cell comprising the nucleic acid or the vector is provided. In one embodiment, the host cell is eukaryotic. In another embodiment, the host cell is selected from a yeast cell, a mammalian cell (e.g., CHO cell or 293 cell) or other cell suitable for the production of antibodies or antigen binding fragments thereof. In another embodiment, the host cell is prokaryotic.

For example, the nucleic acid of the invention comprises:

encoding a polypeptide selected from the group consisting of SEQ ID NOs: 13-22, or encodes a polypeptide that hybridizes to an amino acid sequence set forth in any one of SEQ ID NOs: 13-22, having an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical; or alternatively

Selected from SEQ ID NOs: 39-42, or a nucleic acid selected from the group consisting of SEQ ID NOs: 39-42, has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity.

The invention also encompasses nucleic acids that hybridize under stringent conditions to or have one or more substitutions (e.g., conservative substitutions), deletions, or insertions with: comprising a sequence encoding a polypeptide selected from the group consisting of SEQ ID NOs: 1-9, and a nucleic acid sequence of the amino acid sequence set forth in any one of claims 1-9; or comprises a sequence encoding a sequence selected from the group consisting of SEQ ID NOs: 13-22, having an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical; or comprises a sequence selected from SEQ ID NOs: 39-42, or a nucleic acid comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 39-42, a nucleic acid having a nucleic acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical.

In one embodiment, one or more vectors comprising the nucleic acid are provided. In one embodiment, the vector is an expression vector, such as a eukaryotic expression vector. Vectors include, but are not limited to, viruses, plasmids, cosmids, lambda phage, or Yeast Artificial Chromosomes (YACs). Such as pFuse vectors. Once the expression vector or DNA sequence has been prepared for expression, the expression vector may be transfected or introduced into a suitable host cell. Various techniques may be used to achieve this, such as protoplast fusion, calcium phosphate precipitation, electroporation, retroviral transduction, viral transfection, gene gun, lipid-based transfection, or other conventional techniques. In the case of protoplast fusion, the cells are incubated in medium and screened for appropriate activity. Methods and conditions for culturing the resulting transfected cells and for recovering the resulting antibody molecules are known to those skilled in the art and may be varied or optimized depending on the particular expression vector and mammalian host cell used, based on the present specification and methods known in the art. Alternatively, cells that have stably incorporated DNA into their chromosomes can be selected by introducing one or more markers that allow selection of transfected host cells. The marker may, for example, provide prototrophy, biocidal resistance (e.g., antibiotics) or heavy metal (e.g., copper) resistance to an auxotrophic host, and the like. The selectable marker gene may be directly linked to the DNA sequence to be expressed or introduced into the same cell by co-transformation. Additional elements may also be required for optimal synthesis of mRNA. These elements may include splicing signals, transcriptional promoters, enhancers, and termination signals.

In one embodiment, a host cell comprising one or more polynucleotides of the invention is provided. In some embodiments, host cells comprising the expression vectors of the invention are provided. In some embodiments, the host cell is selected from a yeast cell, a mammalian cell, or other cell suitable for preparing an antibody or antigen-binding fragment thereof.

In one embodiment, the invention provides a method of preparing an antibody molecule of the invention or a fragment thereof (preferably an antigen binding fragment) wherein the method comprises culturing the host cell under conditions suitable for expression of a nucleic acid encoding an antibody molecule of the invention or a fragment thereof (preferably an antigen binding fragment) and optionally isolating the antibody or fragment thereof (preferably an antigen binding fragment). In a certain embodiment, the method further comprises recovering the antibody molecule of the invention or a fragment thereof (preferably an antigen binding fragment) from the host cell.

In one embodiment, a method of making an antibody molecule of the invention is provided, wherein the method comprises culturing a host cell comprising a nucleic acid encoding the antibody (e.g., any one polypeptide chain and/or multiple polypeptide chains) or an expression vector comprising the nucleic acid, as provided above, under conditions suitable for expression of the antibody, and optionally recovering the antibody from the host cell (or host cell culture medium).

For recombinant production of an antibody molecule of the invention, nucleic acids encoding an antibody (e.g., an antibody as described above, e.g., any one polypeptide chain and/or multiple polypeptide chains) are isolated and inserted into one or more vectors for further cloning and/or expression in a host cell. Such nucleic acids are readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of specifically binding to genes encoding heavy and light chains of antibodies).

Antibody molecules prepared as described herein may be purified by known prior art techniques such as high performance liquid chromatography, ion exchange chromatography, gel electrophoresis, affinity chromatography, size exclusion chromatography, and the like. The actual conditions used to purify a particular protein also depend on factors such as net charge, hydrophobicity, hydrophilicity, and the like, and these will be apparent to those skilled in the art. The purity of the antibody molecules of the invention may be determined by any of a variety of well-known analytical methods including size exclusion chromatography, gel electrophoresis, high performance liquid chromatography, and the like.

In some embodiments, the invention also provides methods for identifying, screening, or characterizing the physical/chemical properties and/or biological activity of the antibody molecules of the invention.

In one aspect, antibodies of the invention are tested for antigen binding activity, for example, by known methods such as ELISA, western blotting, and the like. Binding to CD40 can be determined using methods known in the art, exemplary methods being disclosed herein. In some embodiments, a surface plasmon resonance assay (e.g., an affinity measurement) or an ELISA assay is used.

The invention also provides assays for identifying anti-CD 40 antibodies that are biologically active. Biological activity may include, for example, binding to CD40 (e.g., binding to human and/or rhesus CD 40), increasing CD 40-mediated signal transduction (e.g., increasing NFkappa-B signaling pathway), depleting CD 40-expressing cells (e.g., raji or Ramos cells) by directly inducing apoptosis in tumor cells, activating dendritic cells or B cells or T cells (e.g., by increasing cytokine production by T cells), promoting proliferation of T cells (e.g., cd8+ T cells, e.g., activated cd8+ T cells) or B cells, or inhibiting tumor growth.

In certain embodiments, antibodies of the invention are tested for such biological activity.

Activation of T cells ((e.g., cd8+ T cells)) or dendritic cells or B cells can be determined using methods known in the art. For example, by the level (e.g., expression) of the cell activation marker CD8 (T cells) or CD86 (dendritic cells or B cells). Activation of T cells can also be determined using methods well known in the art to determine CD40 signaling (e.g., NF- κb signaling pathway). In one embodiment, transgenic cells are generated that express human CD40 and a reporter gene comprising an NF-kappa B promoter fused to a reporter gene (e.g., beta luciferase, GFP). The addition of anti-CD 40 antibodies to cells results in increased NF-kappa B transcription, which is detected using assays directed against the reporter gene (e.g., luciferase reporter gene assays).

The ADCC effect of an antibody may be determined using methods known in the art. For example by detecting its induction of apoptosis in tumor cells (e.g. by an apoptosis marker molecule such as CD 95), inhibition of tumor cell growth or inhibition of tumor growth in vivo.

Proliferation of T cells (e.g., cd8+ T cells, e.g., activated cd8+ T cells) or B cells can be determined using methods known in the art. Proliferation of B cells is determined, for example, by luminescence methods, such as CellTiter-Glo luminescence. Proliferation of cd8+ T cells is also detected, for example, by OVA-specific OT-I cell methods.

Cells for use in any of the in vitro assays described above include cells that naturally express CD40 or that have been engineered to express CD 40. Such cells include T cells that naturally express CD40L ((e.g., cd8+ T cells, e.g., activated cd8+ T cells)), B cells or dendritic cells that naturally express CD 40. Such cells also include cell lines transfected with CD 40-encoding DNA that expresses CD40 and not normally expresses CD 40.

It will be appreciated that any of the above assays can be performed using the immunoconjugates of the invention in place of or in addition to anti-CD 40 antibodies.

It will be appreciated that any of the above assays can be performed using anti-CD 40 antibodies and other active agents.

In some embodiments, the invention provides pharmaceutical compositions and combinations comprising the antibodies of the invention.

In some embodiments, the invention provides a composition comprising any of the antibody molecules described herein or fragments thereof (preferably antigen binding fragments thereof) or immunoconjugates thereof, preferably the composition is a pharmaceutical composition. In one embodiment, the composition further comprises a pharmaceutical excipient.

The invention also includes compositions (including pharmaceutical compositions or pharmaceutical formulations) comprising an antibody of the invention or an immunoconjugate thereof and/or compositions (including pharmaceutical compositions or pharmaceutical formulations) comprising a polynucleotide encoding an antibody of the invention. In certain embodiments, the compositions comprise one or more antibodies or fragments thereof of the invention or one or more polynucleotides encoding one or more antibodies or fragments thereof of the invention.

These compositions may also contain suitable pharmaceutical excipients, such as pharmaceutically acceptable carriers, pharmaceutically acceptable excipients as known in the art, including buffers.

As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, isotonic and absorption delaying agents, and the like that are physiologically compatible. Pharmaceutically acceptable carriers suitable for use in the present invention may be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. When the pharmaceutical composition is administered intravenously, water is a preferred carrier. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions.

Suitable excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. For the use of excipients and their use, see also "Handbook of PharmaceuticalExcipients", fifth edition, R.C.Rowe, P.J.Seskey and S.C.Owen, pharmaceuticalPress, london, chicago.

The composition may also contain minor amounts of wetting or emulsifying agents, or pH buffering agents, if desired.

The compositions of the present invention may be in a variety of forms. These include, for example, liquid, semi-solid, and solid dosage forms, such as liquid solutions (e.g., injectable solutions and infusible solutions), dispersions or suspensions, liposomal agents, and suppositories. The preferred form depends on the intended mode of administration and the therapeutic use. A common preferred composition is in the form of an injectable solution or an infusible solution. The preferred mode of administration is parenteral (e.g., intravenous, subcutaneous, intraperitoneal (i.p.), intramuscular) injection. In a preferred embodiment, the antibody molecule is administered by intravenous infusion or injection. In another preferred embodiment, the antibody molecule is administered by intramuscular, intraperitoneal or subcutaneous injection.

The agonistic antibodies of the invention that specifically bind human CD40 may be lyophilized for storage and reconstituted in a suitable carrier prior to use. This technique has proven effective for conventional protein formulations and can employ well known lyophilization and reconstitution techniques.

The pharmaceutical compositions or formulations of the present invention may also contain other therapeutic agents as required for the particular indication being treated, preferably those having complementary activities that do not adversely affect each other. Such as chemotherapeutic agents, cytotoxic agents, vaccines, other antibodies, anti-infective agents, small molecule drugs or immunomodulators, and the like.

In some embodiments, the invention also provides a combination product comprising an antibody or antigen-binding fragment thereof of the invention, or an immunoconjugate thereof, and one or more other therapeutic agents (e.g., a chemotherapeutic agent, other antibody, cytotoxic agent, vaccine, anti-infective agent, small molecule drug or immunomodulator, etc.).

In some embodiments, two or more of the ingredients in the combination product may be administered to the subject sequentially, separately or simultaneously in combination.

In some embodiments, the invention also provides kits comprising an antibody, pharmaceutical composition, immunoconjugate or combination of the invention, and optionally a package insert for guiding administration.

In some embodiments, the invention also provides pharmaceutical preparations comprising the antibodies, pharmaceutical compositions, immunoconjugates, combination products of the invention, optionally, the pharmaceutical preparations further comprising a package insert directing administration.

In some embodiments, the other therapeutic agents include, for example, one or more of the following: anti-CTLA-4 antibodies, anti-PD-1 antibodies, anti-PD-L1 antibodies, anti-TIGIT antibodies, anti-OX 40 (also known as CD134, TNFRSF4, ACT35, and/or TXGP 1L) antibodies, anti-LAG-3 antibodies, anti-CD 73 antibodies, anti-CD 137 antibodies, anti-CD 27 antibodies, anti-CSF-1R antibodies, TLR agonists, or small molecule antagonists of IDO or tgfβ. Examples of therapeutic agents that can be combined with the antibodies of the invention are also seen in WO2017/059243 or WO2017/004006.

In some embodiments, the invention also provides a method of enhancing an immune response (e.g., an antigen-specific T cell response) in a subject comprising administering to the subject an effective amount of an anti-CD 40 antibody or antigen-binding fragment thereof of the invention such that the immune response in the individual is enhanced. In some embodiments, the individual has a tumor. In another embodiment, the subject has a viral infection, e.g., a chronic viral infection.

In some embodiments, the invention provides methods of activating T cells (e.g., cd8+ T cells) and/or dendritic cells and/or B cells in a subject using an antibody of the invention, comprising administering to the subject an effective amount of an anti-CD 40 antibody or antigen binding fragment thereof of the invention. The invention also provides methods of promoting proliferation of T cells (e.g., activated cd8+ T cells) or B cells using the methods of the invention, comprising administering to a subject an effective amount of an anti-CD 40 antibody or antigen-binding fragment thereof of the invention. Thus, in some embodiments, given the ability of anti-CD 40 antibodies described herein to enhance co-stimulation of T cell responses, e.g., antigen-specific T cell responses, provided herein are in vitro and in vivo methods of using the antibodies described herein to stimulate, enhance, or up-regulate antigen-specific T cell responses, e.g., anti-tumor T cell responses. Cd4+ and cd8+ T cell responses may be enhanced using anti-CD 40 antibodies. T cells may be T _eff Cells, e.g. CD4+T _eff Cells, CD8+T _eff Cell, T helper (T) _h ) Cell and T toxicity (T) _c ) And (3) cells.

In some embodiments, the invention provides for the use of an antibody molecule of the invention for the treatment or prevention of a disease, such as a tumor or an infection, such as a chronic infection, in which modulation (e.g., enhancement) of an immune response in a subject is desired. Accordingly, the invention also provides a method of inhibiting tumor growth in a subject comprising administering to a subject an anti-CD 40 antibody or antigen-binding fragment thereof of the invention such that tumor growth is inhibited.

In some embodiments, the disease is a CD 40-associated disease, e.g., the disease is a disease of reduced CD40 expression or activity (e.g., as compared to a healthy control), or the disease is a disease of reduced CD40 gene and/or protein levels (e.g., as compared to a healthy control); or the disease benefits from activating CD40 activity, e.g., activating CD40 signaling pathways, and/or activating T cells or B cells or dendritic cells. In some embodiments, the invention relates to a method of activating an antigen activity or activating an antigen-mediated signaling pathway in an individual, the method comprising administering to a subject an effective amount of an antibody or antigen-binding fragment thereof disclosed herein. In some embodiments, activating CD40 activity or activating a CD 40-mediated signaling pathway refers to activating a CD40 signaling pathway.

In some embodiments, the antibodies or antigen binding fragments thereof of the invention are capable of eliciting antibody-dependent cell-mediated cytotoxicity (ADCC), thereby killing tumor cells. Thus, the invention also relates to a method of treating a tumor comprising administering to an individual an effective amount of an antibody or antigen-binding fragment thereof of the invention.

In another aspect, the invention relates to a method of preventing or treating a tumor (e.g., cancer) in a subject, the method comprising administering to the subject an effective amount of an antibody molecule or pharmaceutical composition or immunoconjugate or combination product or kit disclosed herein. In some embodiments, the tumor is a cancer. The tumor may be a solid tumor or a liquid tumor, such as a hematological malignancy. In certain embodiments, the tumor is an immunogenic tumor. In certain embodiments, the tumor is non-immunogenic. In certain embodiments, the tumor is PD-L1 positive. In certain embodiments, the tumor is PD-L1 negative.

In some embodiments, tumors treated and/or prevented with antibody molecules include, but are not limited to, solid tumors, hematological cancers (e.g., leukemia, lymphoma, myeloma, e.g., multiple myeloma), and metastatic lesions. In one embodiment, the cancer is a solid tumor. Examples of solid tumors include malignant tumors, e.g., sarcomas and carcinomas of multiple organ systems, such as those that affect the lung, breast, ovary, lymphoid, gastrointestinal tract (e.g., colon), anus, genitalia, and genitourinary tract (e.g., kidney, bladder epithelium, bladder cells, prostate), pharynx, CNS (e.g., brain, neural, or glial cells), head and neck, skin (e.g., melanoma), nasopharynx (e.g., differentiated or undifferentiated metastatic or locally recurrent nasopharyngeal carcinoma), and pancreatic, as well as adenocarcinomas, including malignant tumors such as colon cancer, rectal cancer, renal cell carcinoma, liver cancer, non-small cell lung cancer, small intestine cancer, and esophageal cancer. The cancer may be in early, intermediate or late stages or metastatic cancer.

In some embodiments, the cancer is selected from colorectal cancer (e.g., CRC), melanoma, e.g., advanced melanoma (e.g., stage II-IV melanoma) or HLA-A2 positive-melanoma; pancreatic cancer, e.g., advanced pancreatic cancer; breast cancer, e.g., metastatic breast cancer or triple negative breast cancer; head and neck cancer (e.g., HNSCC); esophageal cancer; renal Cell Carcinoma (RCC), e.g., renal clear cell carcinoma (ccRCC) or Metastatic Renal Cell Carcinoma (MRCC); lung cancer (e.g., NSCLC); cervical cancer; bladder cancer; or hematological malignancies, e.g., leukemia (e.g., lymphoblastic leukemia) or lymphoma (e.g., hodgkin's Lymphoma (HL), non-hodgkin's lymphoma (NHL), diffuse large B-cell lymphoma (DLBCL), mantle Cell Lymphoma (MCL), or CLL, e.g., recurrent or refractory chronic lymphocytic leukemia).

In some embodiments, examples of cancers further include, but are not limited to, B cell proliferative disorders, which further include, but are not limited to, lymphomas (e.g., B cell non-hodgkin's lymphoma (NHL)) and lymphocytic leukemia.

In some embodiments, the tumor is a tumor, such as a cancer, e.g., a tumor or cancer with T cell dysfunction, that requires activation of T cells or B cells or dendritic cells. In some embodiments, the tumor is a tumor in which expression or activity of OX40 is reduced. In some embodiments, the tumor is a tumor that benefits from activation of OX40 signaling pathway, e.g., cancer.

In some embodiments, the cancer described herein is lymphoma, colon cancer, colorectal cancer, rectal cancer, lung cancer (e.g., non-small cell lung cancer), liver cancer, stomach cancer, and metastatic cancers thereof.

In some embodiments, the antibodies or antigen binding fragments thereof of the invention are not suitable for treating hematological cancers with CD40 expression, which may be exacerbated by treatment with CD40 agonists. Some cancers may be known to express CD40 and thus experience such exacerbations and thus may be excluded from the category. In specific embodiments, specific tumor samples are tested for CD40 expression and excluded from therapy with a CD40 antibody of the invention based on the test results.

The methods and compositions disclosed herein are useful for treating metastatic lesions associated with the aforementioned cancers.

The antibodies or antigen-binding fragments thereof of the invention that specifically bind human CD40 may also be administered prophylactically to reduce the risk of developing cancer, delay the onset of an event in the progression of cancer, and/or reduce the risk of recurrence after cancer remission. This may be particularly useful for patients where the tumor is difficult to locate and known to have a tumor due to other biological factors.

In another aspect, the invention relates to a method of preventing or treating an infectious disease in a subject, the method comprising administering to the subject an effective amount of an antibody molecule or pharmaceutical composition or immunoconjugate or combination product or kit disclosed herein. In one embodiment, the infectious disease is chronic infection. In some embodiments, the infection is a viral infection.

In some embodiments, the infection is acute or chronic. In some embodiments, the chronic infection is a persistent infection, a latent infection, or a slow infection. In some embodiments, the chronic infection is caused by a pathogen selected from the group consisting of bacteria, viruses, fungi, and protozoa.

In some embodiments, some examples of viruses include HIV, hepatitis (hepatitis a, hepatitis b, and hepatitis c), herpes viruses (e.g., VZV, HSV-1, HAV-6, HSV-II, and CMV, EB virus), adenoviruses, influenza viruses, flaviviruses, epox viruses, rhinoviruses, coxsackie viruses, coronaviruses, respiratory syncytial viruses, mumps viruses, rotaviruses, measles viruses, rubella viruses, parvoviruses, vaccinia viruses, HTLV viruses, dengue viruses, papillomaviruses, molluscum viruses, polio viruses, rabies viruses, JC viruses, and arboencephalitis viruses; some examples of bacteria include chlamydia, rickettsia, mycobacteria, staphylococci, streptococci, pneumococci, meningococci and gonococci, klebsiella, proteus, serratia, pseudomonas, legionella, diphtheria, salmonella, bacillus, cholera, tetanus, botulism, anthrax, plague, leptospirosis and lyme disease pathogens; some examples of fungi include candida (candida albicans, candida krusei, candida glabrata, candida tropicalis, etc.), cryptococcus neoformans (Cryptococcus neoformans), aspergillus (aspergillus fumigatus, aspergillus niger, etc.), mucor orders (mucor, colubus, rhizopus), mycelial chocola (Sporothrix schenkii), blastomyces dermatitis (Blastomyces dermatitidis), paracoccidiomycetes brazil (Paracoccidioides brasiliensis), paracoccidiomycetes crudus (Coccidioides immitis), and histoplasma capsulatum (Histoplasma capsulatum); some examples of parasites include amoeba dysenteriae (Entamoeba histolytica), ciliate colonospora (balntidium coll), british amoeba (naegleriafawnri), acanthamoeba sp, giardia lamblia sp, cryptosporidium sp, pneumosporidium californicum Pneumocystis carinii, plasmodium vivax Plasmod ium viva x, babesia minutissima (Ba besia microti), trypanosoma brucei Try pa nosomabrucei, trypanosoma cruzi Trypanosoma cruzi, leishmania donovani Leishmania donovani, toxoplasma just Toxoplasma gondii, and caenorhabditis brasiliensis Nippostrongylus brasiliensis.

In some embodiments, the infectious disease treated and/or prevented with the antibody molecule includes pathogens for which no effective vaccine is currently available or for which conventional vaccines are not yet fully effective. These include, but are not limited to, HIV, (type a, type b and type c) hepatitis, influenza, herpes, giardia (Giardia), malaria, leishmania (Leishmania), staphylococcus aureus (Staphylococcus aureus), pseudomonas aeruginosa (Pseudomonas aeruginosa).

Diseases that can be treated with the methods and compositions disclosed herein are also described in WO2017/059243 or WO2017/004006.

In some embodiments, the antibodies or pharmaceutical compositions or immunoconjugates or combination products or kits of the invention can also be administered in combination with one or more other therapies, e.g., therapeutic modalities and/or other therapeutic agents, for prophylaxis and/or treatment as described herein.

In some embodiments, the treatment modality includes surgery (e.g., tumor resection); radiation therapy (e.g., external particle beam therapy, which involves three-dimensional conformal radiation therapy in which the irradiation region is designed), localized irradiation (e.g., irradiation directed at a preselected target or organ), or focused irradiation), and the like. The focused radiation may be selected from stereotactic radiosurgery, split stereotactic radiosurgery, and intensity modulated radiation therapy. The focused radiation may have a radiation source selected from the group consisting of a particle beam (proton), cobalt-60 (photon) and a linear accelerator (X-ray). Radiation therapy may be administered by one or a combination of several methods including, but not limited to, external particle beam therapy, internal radiation therapy, implant irradiation, stereotactic radiation surgery, whole body radiation therapy, and permanent or brief interstitial brachytherapy.

In some embodiments, the therapeutic agent is selected from a chemotherapeutic agent, a cytotoxic agent, a vaccine, other antibodies, an anti-infective agent, or an immunomodulatory agent (e.g., an activator of a costimulatory molecule or an inhibitor of an immune checkpoint molecule).

Exemplary other antibodies include, but are not limited to, immune checkpoint inhibitors (e.g., anti-CTLA-4, anti-TIM-3, anti-CEACAM); antibodies that stimulate immune cells (e.g., agonistic GITR antibodies or CD137 antibodies); anti-cancer antibodies (e.g., rituximab @Or->) Trastuzumab->Toximomab->Tilmimumab->Alemtuzumab->Epalzumab ∈ ->Bevacizumab->Erlotinib>Cetuximab->Etc. For example, the other antibody may be an anti-PD-L1 antibody, an anti-LAG-3 antibody, an anti-PD-1 antibody or an anti-CLA-4 antibody.

Exemplary vaccines include, but are not limited to, cancer vaccines. The vaccine may be a DNA-based vaccine, an RNA-based vaccine or a virus-transduction based vaccine. Cancer vaccines can be prophylactic or therapeutic, such as cancer cells, purified tumor antigens (including recombinant proteins, peptides and carbohydrate molecules), cells, and cells transfected with genes encoding immunostimulatory cytokines (He et al (2004) J.Immunol.173:4919-28). Non-limiting examples of tumor vaccines that can be used include peptides of melanoma antigens, such as peptides of gp100, MAGE antigens, trp-2, MART1 and/or tyrosinase, or tumor cells transfected to express the cytokine GM-CSF. In some embodiments, the cancer vaccine is a peptide cancer vaccine, which in some embodiments is a personalized peptide vaccine. In some embodiments, the peptide Cancer vaccine is a multivalent long peptide, a multiple peptide, a peptide mixture, a hybrid peptide, or a peptide pulsed dendritic cell vaccine (see, e.g., yamada et al, cancer Sci,104:14-21, 2013). In some embodiments of any of the methods of the invention, the administration of the antibodies or fragments thereof of the invention is combined with the administration of a tumor antigen. The antigen may be, for example, a tumor antigen, a viral antigen, a bacterial antigen or an antigen from a pathogen. In some embodiments, the tumor antigen comprises a protein. In some embodiments, the tumor antigen comprises a nucleic acid. In some embodiments, the tumor antigen is a tumor cell.

Exemplary anti-infective agents include, but are not limited to, antiviral agents, antifungal agents, antiprotozoal agents, antibacterial agents, such as the nucleoside analogs zidovudine (AST), ganciclovir, foscarnet, or cidovir, among others.

Immunomodulators include immune checkpoint molecule inhibitors and costimulatory molecule activators.

In some embodiments, the inhibitor of an immune checkpoint molecule is an inhibitor of CTLA-4, TIM-3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, CEACAM (e.g., CEACAM-1 and/or CEACAM-5) and/or TGFR. Inhibition of the molecule may be at the DNA, RNA or protein level. In some embodiments, an inhibitory nucleic acid (e.g., dsRNA, siRNA or shRNA) can be used to inhibit expression of an immune checkpoint molecule. In other embodiments, the inhibitor of the immune checkpoint molecule is a polypeptide, e.g., a soluble ligand or an antibody or antibody fragment, that binds to the immune checkpoint molecule.

In some embodiments, the immunomodulator is an activator or agonist of a costimulatory molecule. In one embodiment, the agonist of the co-stimulatory molecule is selected from the group consisting of agonists of the following molecules (e.g., agonistic antibodies or antigen binding fragments thereof, or soluble fusions): OX40, CD2, CD27, CDS, ICAM-1, LFA-1 (CD 11a/CD 18), ICOS (CD 278), 4-1BB (CD 137), GITR, CD30, CD40, BAFFR, HVEM, CD7, LIGHT, NKG2C, SLAMF7, NKp80, CD160, B7-H3 or CD83 ligand.

In some embodiments, the antibodies or fragments thereof of the invention can be administered in combination with a treatment comprising adoptively transferring T cells (e.g., cytotoxic T cells or CTLs) expressing a Chimeric Antigen Receptor (CAR).

In some embodiments, the antibodies or fragments thereof of the invention may be administered in combination with an anti-neoplastic agent or oncolytic virus.

In all of the above methods, CD40 agonism may be combined with other forms of immunotherapy such as cytokine therapy (e.g., interferon, GM-CSF, G-CSF, IL-2) or bispecific antibody therapy, which provides enhanced tumor antigen presentation. See, e.g., holliger (1993) proc.Natl. Acad. Sci. USA 90:6444-6448; poljak (1994) Structure2:1121-1123.

In some embodiments, the antibodies or fragments thereof of the invention may be combined with conventional methods of enhancing immune function in a host, including, but not limited to: (i) APC enhancement such as (a) injection of DNA encoding heterologous MHC alloantigen into the tumor, or (B) transfection of biopsied tumor cells with genes that increase the likelihood of immune antigen recognition (e.g., immunostimulatory cytokines, GM-CSF, co-stimulatory molecules B7.1, B7.2), (iii) adoptive cell immunotherapy, or treatment with activated tumor-specific T cells. Adoptive cellular immunotherapy involves isolating tumor-infiltrating host T lymphocytes, such as stimulating in vitro expansion of the population by IL-2 or tumor or both; alternatively, dysfunctional isolated T cells may be activated by in vitro application of the antibodies of the invention, and the T cells so activated may then be re-administered to a host.

The various combination therapies described above may be further combined for treatment.

Further examples of combinations of antibodies of the invention with other therapeutic modalities or agents can be found in WO2017/059243 or WO2017/004006, etc.

Such combination therapies encompass the administration of a combination (wherein two or more therapeutic agents are contained in the same formulation or separate formulations), and the administration of a separate, in which case the administration of an antibody of the invention may occur prior to, concurrent with, and/or subsequent to the administration of another therapy, e.g., a therapeutic regimen and/or therapeutic agent. The antibody molecule and/or other therapy, e.g., therapeutic agent or mode of treatment, may be administered during active disease or during remission or less active disease. The antibody molecule may be administered prior to, concurrently with, after, or during remission of the disease.

It will be appreciated that any treatment can be performed using the immunoconjugate or composition or combination product or kit of the invention in place of or in addition to the antibody of the invention.

The mode of administration of the antibodies (and pharmaceutical compositions or immunoconjugates comprising the same, as well as any additional therapeutic agents) of the invention may be any suitable route, such as parenteral administration, e.g., intradermal, intramuscular, intraperitoneal, intravenous or subcutaneous, mucosal (oral, intranasal, intravaginal, rectal) or other means as known to those of skill in the art. Agonistic antibodies that specifically bind CD40 can be intratumorally administered to lymph node drainage sites for local delivery into tumors using known methods. The agonistic antibodies of the invention that specifically bind to human CD40 may be administered to a patient by any suitable route, for example parenterally by intravenous (i.v.) infusion or bolus injection, intramuscular or subcutaneous or intraperitoneal injection.

Various dosing schedules are contemplated herein, including, but not limited to, single dosing or multiple dosing at multiple time points, bolus dosing, and pulse infusion.

For the prevention or treatment of a disease, the appropriate dosage of the antibodies of the invention (when used alone or in combination with one or more other therapeutic agents) will depend on the type of disease to be treated, the type of antibody, the severity and course of the disease, whether the antibody is administered for prophylactic or therapeutic purposes, previous treatments, the patient's clinical history and response to the antibody, and the discretion of the attending physician. The antibody is suitably administered to the patient in one treatment or over a series of treatments. Typically, the clinician administers the composition until a dose is reached that achieves the desired effect. The antibodies of the invention may thus be administered in a single dose, or in two or more doses (which may contain the same or different amounts of the desired molecule) over a period of time, or by continuous infusion through an implant device or catheter. The appropriate dose may be determined by using appropriate dose response data. In certain embodiments, the antibody may be administered to the patient over an extended period of time. In certain embodiments, the antibody is administered weekly, biweekly, monthly, bi-monthly, tri-monthly, tetra-monthly, penta-monthly, or hexa-monthly.

In certain embodiments, the anti-CD 40 antibodies, or antigen-binding fragments thereof, provided herein may be used to detect the presence of CD40 in a biological sample. The term "detection" as used herein, including quantitative or qualitative detection, exemplary detection methods may involve immunohistochemistry, immunocytochemistry, flow cytometry (e.g., FACS), magnetic beads complexed with antibody molecules, ELISA assays, PCR-techniques (e.g., RT-PCR). In certain embodiments, the biological sample is blood, serum, or other liquid sample of biological origin. In certain embodiments, the biological sample comprises a cell or tissue. In some embodiments, the biological sample is from a lesion associated with a disease (e.g., a tumor or infection) described herein

In some embodiments, CD40 is human CD40 or cynomolgus monkey CD40. In some embodiments, the method comprises contacting the biological sample with an anti-CD 40 antibody or antigen-binding fragment thereof as described herein under conditions that allow the anti-CD 40 antibody to bind to CD40, and detecting whether a complex is formed between the anti-CD 40 antibody and CD40. The formation of a complex indicates the presence of CD40. The method may be an in vitro or an in vivo method. In one embodiment, an anti-CD 40 antibody is used to select a subject suitable for treatment with the anti-CD 40 antibody, e.g., wherein CD40 is a biomarker for selecting the subject.

In one embodiment, the antibodies of the invention can be used to diagnose a disease described herein, e.g., to evaluate (e.g., monitor) the treatment or progression of a disease described herein, diagnosis and/or staging thereof in a subject. In certain embodiments, a labeled anti-CD 40 antibody is provided. Labels include, but are not limited to, labels or moieties that are detected directly (e.g., fluorescent labels, chromophore labels, electron dense labels, chemiluminescent labels, and radiolabels), as well as moieties that are detected indirectly, such as enzymes or ligands, e.g., by enzymatic reactions or molecular interactions. Exemplary labels include, but are not limited to, radioisotopes ³² P、 ¹⁴ C、 ¹²⁵ I、 ³ H and ¹³¹ i, fluorophores such as rare earth chelates or luciferins and derivatives thereof, rhodamine and derivatives thereof, dansyl, umbelliferone, luciferases (luciferases), e.g., firefly luciferases and bacterial luciferases (U.S. Pat. No. 4,737,456), luciferins, 2, 3-dihydrophthalazinediones, horseradish peroxidase (HR),alkaline phosphatase, beta-galactosidase, glucoamylase, lytic enzymes, carbohydrate oxidase, e.g., glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase, heterocyclic oxidases such as uricase and xanthine oxidase, and enzymes that oxidize dye precursors using hydrogen peroxide such as HR, lactoperoxidase, or microperoxidase (microperoxidase), biotin/avidin, spin-labeling, phage labeling, stable free radicals, and the like.

In some embodiments of any of the inventions provided herein, the sample is obtained prior to treatment with the anti-CD 40 antibody. In some embodiments, the sample is obtained prior to treatment with a disease drug described herein. In some embodiments, the sample is formalin fixed, paraffin coated (FFPE). In some embodiments, the sample is a biopsy (e.g., core biopsy), a surgical specimen (e.g., a specimen from a surgical resection), or a fine needle aspirate.

In some embodiments, CD40 is detected prior to treatment, e.g., prior to initiation of treatment or prior to a certain treatment after a treatment interval.

In some embodiments, provided are test kits comprising an antibody or antigen binding fragment thereof of the invention for diagnosing a disease described herein, such as a tumor or infection.

In some embodiments, there is provided a method of treating a disease described herein, such as a tumor or infection, comprising: a subject (e.g., a sample) (e.g., a subject sample) is tested for the presence of CD40, thereby determining a CD40 value, the CD40 value is compared to a control value, and if the CD40 value is less than the control value, a therapeutically effective amount of an anti-CD 40 antibody (e.g., an anti-CD 40 antibody described herein), optionally in combination with one or more other therapies, is administered to the subject, thereby treating a disease described herein, such as a tumor or infection.

The invention thus also relates to the use of an antibody or antigen-binding fragment thereof of the invention for the above-described methods, as well as to the use of an antibody or antigen-binding fragment thereof of the invention for the manufacture of a medicament or composition or combination or kit for the above-described methods, and/or the use of an antibody or antigen-binding fragment thereof of the invention for the manufacture of a kit for the diagnosis of a disease as described herein.

Methods and uses of antibodies or antigen-binding fragments thereof suitable for use in the present invention are also applicable to immunoconjugates, compositions, combinations, or kits comprising the antibodies or antigen-binding fragments thereof of the present invention.

The invention is further illustrated in the following figures. However, these drawings and specific embodiments of the present invention should not be construed as limiting the scope of the present invention, and variations that would be apparent to one skilled in the art are intended to be included within the spirit of the present invention and the scope of the appended claims.

Drawings

FIG. 1 shows the monoclonal activation of Jurkat/NF-. Kappa.B-GFP+hCD 40 reporter cells by HEL and CD40L produced by 293F cells as determined by flow cytometry, with GFP expression following activation of the reporter cells, GFP positive rates being detected by FITC channels.

FIG. 2 shows the positive rate of CD40 binding antibodies in the antibody library obtained by phage ELISA assay of the second round (FIG. 2A) and third round (FIG. 2B) of phage display enrichment, and antibodies with a binding signal to CD40 greater than three times greater than the binding signal to BSA were defined as positive antibodies.

FIG. 3 shows the activation of Jurkat/NF- κB-GFP+hCD40 reporter cells by supernatant containing negative control N27, positive CD40 agonist antibodies produced by monoclonal measurement of Jurkat/NF- κB-GFP+hCD40 reporter cells by flow cytometry. The fluorescence intensity of GFP detected by FITC channel represents the activation degree of the monoclonal.

FIG. 4 shows the NK003 multimeric properties of the antibodies of the invention produced by 293F cells as determined by size exclusion chromatography.

FIG. 5A shows that the antibody NK003 of the present invention produced by 293F cells specifically binds to CD40, did not bind or did not bind to other TNFR family members OX40, 4-1BB, GITR, as determined by ELISA. FIG. 5B shows the specific binding of the antibody NK004 of the present invention to CD40 by ELISA assay of 293F cells.

FIG. 6 shows the competitive binding of the antibody NK003 of the present invention to CD40L by ELISA assay 293F cells to CD40.

FIG. 7A shows that the antibody NK003 of the present invention produced by 293F cells was assayed by flow cytometry to activate Jurkat/NF-. Kappa.B-GFP+hCD 40 reporter cells in a crosslinked form, and FIG. 7B shows that the antibody NK004 of the present invention produced by 293F cells was assayed by flow cytometry to activate Jurkat/NF-. Kappa.B-GFP+hCD 40 reporter cells in a constitutive form independent of crosslinking. FITC channel detects GFP, MFI is defined as the product of geometric Mean of GFP positive cells and the percentage of GFP positive cells.

FIG. 8 shows the binding of the NK003 profile of the present invention produced by 293F cells to 293FT cells overexpressing human CD40 (FIG. 8A) and 293FT cells overexpressing rhesus CD40 (FIG. 8B) as determined by flow cytometry.

FIG. 9 shows that NK003, an antibody of the present invention produced by 293F cells, induces apoptosis of Raji cells (FIG. 9A) and Ramos cells (FIG. 9B) as measured by flow cytometry, and the apoptosis index is CD95 expression. MFI is defined as the product of the geometric mean of CD95 positive cells and the percentage of CD95 positive cells.

FIG. 10 shows the activation of dendritic cells in PBMC by the antibody NK003 of the present invention produced by 293F cells as determined by flow cytometry in the absence of crosslinker (FIG. 10A) and in the presence of crosslinker anti-Fc (FIG. 10B), respectively. The index of dendritic cell activation is the expression of CD 86. MFI is defined as the product of the geometric mean of CD86 positive cells and the percentage of CD86 positive cells.

FIG. 11 shows the proliferation of B cells in PBMC by the antibody NK003 of the present invention produced by 293F cells as measured by CellTiter-Glo method, the cell proliferation index being fluorescence intensity (RLU), a larger RLU value indicating more cells.

FIG. 12 shows the activation of B cells in PBMC by the antibody NK003 of the invention produced by 293F cells as determined by flow cytometry, without the addition of cross-linking agent (FIG. 12A) and with the addition of cross-linking agent anti-Fc (FIG. 12B), respectively. The B cell activation index is CD86 expression. MFI is defined as the product of the geometric mean of CD86 positive cells and the percentage of CD86 positive cells.

FIG. 13 shows tumor growth curves (FIG. 13A) and survival statistics (FIG. 13B) of individual mice, when Raii cells are inoculated in SCID mice, and the antibody NK003 of the present invention and negative control HEL produced in 293F cells are administered simultaneously.

FIG. 14 shows the activation of the immune system by administration of the antibody NK003 of the invention and the negative control HEL produced in 293F cells, OT1 cell number, OT1 cell to CD8 cell ratio (OT 1/CD8 ⁺ Ratio) and an increase in the ratio of CD8 cells to CD4 cells (CD 8/CD4 ratio) are indicative of an activation of the immune system.

FIG. 15 shows the tumor growth curves (FIG. 15A) and the body weight change curves (FIG. 15B) of individual mice, given the antibodies NK003, negative control HEL and positive control CP870893 of the invention produced in 293F cells in MC38 tumor-bearing CD40 humanized mice.

FIG. 16 shows activation of Jurkat/NF-. Kappa.B-GFP+hCD 40 reporter cells by flow cytometry assayed by 293F cells producing antibodies of the invention NK003, fcgammaRIIB enhanced mutant NK003-V12, fcgammaRIIA/FcgammaRIIB enhanced mutant NK003-S267E/L328F, and negative control HEL, respectively, under crosslinking of 293 FT-FcgammaRIIA cells (FIG. 16A) and 293 FT-FcgammaRIIB cells (FIG. 16B). FITC channel detects GFP, MFI is defined as the product of the geometric mean of GFP-positive cells and the percentage of GFP-positive cells.

FIG. 17 shows a plasmid map of NK003 inserted as Fab into pcomb3 vector.

FIG. 18 shows the positive rate of CD40 binding antibodies in the antibody library obtained by the third round of phage display as determined by phage ELISA, with antibodies that bind CD40 more than three times greater than the binding signal to BSA defined as positive antibodies.

FIG. 19 shows NK003 VH (FIG. 19A), VL (FIG. 19B) complementarity determining region CDR3 affinity maturation three generation sequencing results.

FIG. 20 shows the activation of Jurkat/NF-. Kappa.B-GFP+hCD 40 reporter cells in cross-linked form by flow cytometry for NK003-AM-9, NK003-AM-18, antibodies of the invention produced by 293F cells, the GFP being detected by the FITC channel, MFI being defined as the product of the geometric Mean of GFP positive cells and the percentage of GFP positive cells.

FIG. 21 shows NK003 VH (FIG. 21A), VL (FIG. 21B) framework region affinity maturation three-generation sequencing results.

FIG. 22 shows the activation of Jurkat/NF-. Kappa.B-GFP+hCD 40 reporter cells in cross-linked form by flow cytometry for the antibody NK003-AM-18-EP1 of the invention produced by 293F cells, the GFP being detected by the FITC channel, MFI being defined as the product of the geometric Mean of GFP positive cells and the percentage of GFP positive cells.

Definition of the definition

It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

For purposes of explaining the present specification, the following definitions will be used, and terms used in the singular form may also include the plural, and vice versa, as appropriate. It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

The term "about" when used in conjunction with a numerical value is intended to encompass numerical values within a range having a lower limit of 5% less than the specified numerical value and an upper limit of 5% greater than the specified numerical value.

As used herein, the term "and/or" means any one of the selectable items or two or more of the selectable items.

As used herein, the terms "comprises" or "comprising" are intended to include the stated elements, integers or steps but do not exclude any other elements, integers or steps. In this document, the terms "comprises" or "comprising" when used herein, unless otherwise indicated, also encompass the instances of the recited elements, integers, or steps in combination. For example, when referring to an antibody variable region "comprising" a particular sequence, it is also intended to encompass antibody variable regions consisting of that particular sequence.

As described herein, the term "CD40" is known in the art, such as human CD40 or rhesus CD40.CD40 is also known as tumor necrosis factor receptor superfamily member 5 (TNFRSF 5), CD4OL receptor, or CD154 receptor. The human full length CD40 protein is a type I membrane protein with 277 amino acids, see for example NCBI, nm_001250.5. Rhesus CD40 is described, for example, in NCBI, nm_001265862.1.

The terms "anti-CD 40 antibody", "anti-CD 40", "CD40 antibody" or "antibody that binds CD40" or "antibody that specifically binds CD40" as used herein refer to an antibody that is capable of binding (human or rhesus) CD40 subunits or fragments thereof with sufficient avidity such that the antibody may be used as a diagnostic and/or therapeutic agent in targeting (human or rhesus) CD 40. In one embodiment, the extent of binding of an anti-CD 40 antibody to a non-human or rhesus CD40 protein is less than about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80% or about 90% or more of the binding of the antibody to a (human or rhesus) CD40, as measured, for example, by Radioimmunoassay (RIA) or biological optical interferometry or MSD assay.

Antibodies that specifically bind human CD40 may be cross-reactive to other related antigens, e.g., to the same antigen from other species (homologous), such as rhesus monkeys. Although monospecific antibodies specifically bind one antigen or one epitope, bispecific antibodies specifically bind two different antigens or two different epitopes.

"complementarity determining regions" or "CDR regions" or "CDRs" are regions of an antibody variable domain that are hypervariable in sequence and form structurally defined loops ("hypervariable loops") and/or contain antigen-contacting residues ("antigen-contacting points"). CDRs are mainly responsible for binding to the epitope. CDRs of the heavy and light chains are commonly referred to as CDR1, CDR2, and CDR3, numbered sequentially from the N-terminus. CDRs located within the antibody heavy chain variable domain are referred to as HCDR1, HCDR2 and HCDR3, while CDRs located within the antibody light chain variable domain are referred to as LCDR1, LCDR2 and LCDR3. In a given light chain variable region or heavy chain variable region amino acid sequence, the exact amino acid sequence boundaries of each CDR can be determined using any one or a combination of a number of well-known antibody CDR assignment systems, including, for example: chothia (Chothia et al, (1989) Nature 342:877-883, al-Lazikani et al, "Standard conformations for the canonical structures of immunoglobulins", journal of Molecular Biology,273, 927-948 (1997)) based on the three-dimensional structure of antibodies and topology of CDR loops, kabat (Kabat et al, sequences of Proteins of Immunological Interest, 4 th edition, U.S. Pat. No. of Health and Human Services, national Institutes of Health (1987)), abM (University of Bath), contact (University College London), international ImMunoGeneTics database (IMGT) (on the world Wide Web) based on neighbor-transmitted clusters (CDR affinity propagation clustering) using a large number of crystal structures.

For example, the residues of each CDR are as follows, according to different CDR determination schemes.

CDRs may also be determined based on having the same Kabat numbering positions as the reference CDR sequences (e.g., any of the exemplary CDRs of the invention).

In the present invention, unless otherwise indicated, the term "CDR" or "CDR sequence" encompasses CDR sequences determined in any of the above-described ways.

In the present invention, unless otherwise indicated, when referring to residue positions in the antibody variable region, including heavy chain variable region residues and light chain variable region residues, reference is made to numbering positions according to the Kabat numbering system (Kabat et al Sequences of Proteins of Immunological Interest,5th Ed.Public Health Service,National Institutes of Health,Bethesda,Md. (1991)).

In one embodiment, the CDRs of the antibodies of the invention are bordered by IMGT rules, e.g., using IMGT databases.

It should be noted that the boundaries of CDRs of variable regions of the same antibody obtained based on different assignment systems may differ. I.e. the CDR sequences of the same antibody variable region defined under different assignment systems are different. Thus, when referring to defining antibodies with a particular CDR sequence as defined herein, the scope of the antibodies also encompasses antibodies whose variable region sequences comprise the particular CDR sequence, but whose purported CDR boundaries differ from the particular CDR boundaries defined herein by the application of different protocols (e.g., different assignment system rules or combinations).

"Fc region" or "Fc domain" or "Fc" refers to the C-terminal region of the heavy chain of an antibody that mediates binding of an immunoglobulin to host tissues or factors, including binding to Fc receptors located on various cells of the immune system (e.g., effector cells) or binding to the first component of the classical complement system (C1 q). Thus, the Fc region comprises the constant region of an antibody that excludes the first constant region immunoglobulin domain (e.g., CH1 or CL). In IgG, igA, and IgD isotypes, the Fc region comprises CH2 and CH3 constant regions in each of the two heavy chains of the antibody; igM and IgE Fc regions contain three heavy chain constant domains (CH domains 2-4) in each polypeptide chain. For IgG, the Fc region comprises immunoglobulin domains cγ2 and cγ3 and a hinge between cγ1 and cγ2. Although the boundaries of the Fc region of an immunoglobulin heavy chain may vary, a human IgG heavy chain Fc region is generally defined as a fragment from an amino acid residue at position C226 or P230 (or an amino acid between these two amino acids) to the carboxy-terminus of the heavy chain, with numbering according to the EU index in Kabat. Kabat et al (1991) Sequences of Proteinsof Immunological Interest, national Institutes of Health, bethesda, MD; see also figures 3c-3f of U.S. patent application publication No. 2008/02480228. The CH2 domain of the human IgG Fc region extends from about amino acid 231 to about amino acid 340, while the CH3 domain is located on the C-terminal side of the CH2 domain in the Fc region, i.e., it extends from about amino acid 341 to about amino acid 447 (including the C-terminal lysine) of the IgG. As used herein, an Fc region may be a native sequence Fc, including any allotypic variant, or variant Fc (e.g., a non-naturally occurring Fc). Fc may also refer to this region in isolation or in the context of an Fc-containing protein polypeptide such as a "binding protein comprising an Fc region" also known as an "Fc fusion protein" (e.g., an antibody or immunoadhesin).

As used herein, the term "epitope" refers to the portion of an antigen (e.g., CD 40) that specifically interacts with an antibody molecule. Epitopes within a protein antigen may be formed from contiguous amino acids (typically linear epitopes) or discrete amino acids juxtaposed by tertiary folding of the protein (typically conformational epitopes). Epitopes formed by consecutive amino acids typically (but not always) remain exposed to denaturing solvents, whereas epitopes formed by tertiary folding are typically lost when treated with denaturing solvents. Epitopes generally comprise at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids in a unique spatial conformation.

An "antibody that binds to the same or overlapping epitope" as a reference antibody refers to an antibody that blocks 50%, 60%, 70%, 80%, 90% or 95% or more of the binding of the reference antibody to its antigen in a competition assay, and conversely, a reference antibody blocks 50%, 60%, 70%, 80%, 90% or 95% or more of the binding of the antibody to its antigen in a competition assay.

An antibody that competes with a reference antibody for binding to its antigen refers to an antibody that blocks 50%, 60%, 70%, 80%, 90% or more than 95% of the binding of the reference antibody to its antigen in a competition assay. In contrast, the reference antibody blocks 50%, 60%, 70%, 80%, 90% or 95% or more of the binding of the antibody to its antigen in a competition assay. Numerous types of competitive binding assays can be used to determine whether an antibody competes with another, such as: solid phase direct or indirect Radioimmunoassay (RIA), solid phase direct or indirect Enzyme Immunoassay (EIA), sandwich competition assay (see, e.g., stahli et al, 1983,Methods in Enzymology 9:242-253).

An antibody that inhibits (e.g., competitively inhibits) binding of a reference antibody to its antigen refers to an antibody that inhibits 50%, 60%, 70%, 80%, 90% or more than 95% of binding of the reference antibody to its antigen. Conversely, a reference antibody inhibits the binding of 50%, 60%, 70%, 80%, 90% or 95% or more of the antibody to its antigen. The binding of an antibody to its antigen can be measured by affinity (e.g., equilibrium dissociation constant). Methods for determining affinity are known in the art.

An antibody that exhibits the same or similar binding affinity and/or specificity as a reference antibody refers to an antibody that is capable of having at least 50%, 60%, 70%, 80%, 90% or more than 95% of the binding affinity and/or specificity of the reference antibody. This can be determined by any method known in the art for determining binding affinity and/or specificity.

An "antibody of the IgG form" refers to an IgG form to which the heavy chain constant region of an antibody belongs. The heavy chain constant regions are identical for all of the same type of antibody and differ between different types of antibodies. For example, an antibody in the form of an IgG1 refers to an Ig domain whose heavy chain constant region Ig domain is an IgG1 domain.

"human" antibody (HuMAb) refers to an antibody having variable regions in which both framework and CDR regions are derived from human germline immunoglobulin sequences. Furthermore, if the antibody contains constant regions, the constant regions are also derived from human germline immunoglobulin sequences.

"humanized" antibodies refer to antibodies in which some, most, or all of the amino acids outside of the CDR domains of a non-human antibody (e.g., a mouse antibody) have been replaced with the corresponding amino acids derived from a human immunoglobulin. In one embodiment of the humanized form of an antibody, some, most or all of the amino acids outside of the CDR domains have been replaced with amino acids from a human immunoglobulin, while some, most or all of the amino acids within one or more CDR regions have not been altered. Small additions, deletions, insertions, substitutions or modifications of amino acids are permissible provided they do not eliminate the ability of an antibody to bind to a particular antigen. "humanized" antibodies retain antigen specificity similar to the original antibody.

As used herein, "chimeric antibody" refers to an antibody in which the variable region is derived from one species and the constant region is derived from another species, such as an antibody in which the variable region is derived from a mouse antibody and the constant region is derived from a human antibody.

As used herein, "antibody fragment" refers to a fragment that differs from an intact antibodyComprising a portion of an intact antibody and binding to an antigen to which the intact antibody binds. As used herein, the term "antigen-binding fragment" as used herein refers to one or more fragments of an antibody that retain the ability to specifically bind an antigen (e.g., human CD 40). Examples of antibody fragments include, but are not limited to Fv, fab, fab ', fab ' -SH, F (ab ') ₂ The method comprises the steps of carrying out a first treatment on the surface of the diabody; a linear antibody; single chain antibodies (e.g., scFv); single domain antibodies; a bivalent or bispecific antibody or fragment thereof; camelidae antibodies; and bispecific or multispecific antibodies formed from antibody fragments.

As used herein, "multispecific" refers to an antibody that specifically binds to at least two different antigens or two different epitopes within an antigen, e.g., three, four, or five different antigens or epitopes.

As used herein, "bispecific" refers to antibodies that specifically bind to two different antigens or two different epitopes within the same antigen. Bispecific antibodies may be cross-reactive with other related antigens or may bind epitopes shared between two or more different antigens.

As used herein, the term "cross-linking" refers to the higher multimerization of CD40 on cells induced by the binding of an antibody that specifically binds human CD40 to cis or trans fcyriib, resulting in the induction of agonistic activity of CD 40. The cross-linking can be assessed in vitro using anti-human F (ab') 2 as described herein as a cross-linking agent.

As used herein, the term "cross-reactive" refers to the ability of an antibody described herein to bind CD40 from a different species. For example, an antibody described herein that binds human CD40 also binds CD40 from another species (e.g., cynomolgus monkey CD 40). As used herein, cross-reactivity may be measured by detecting specific reactivity or binding or otherwise functionally interacting with cells physiologically expressing CD40 with purified antigen in a binding assay (e.g., SPR, ELISA). Methods for determining cross-reactivity include standard binding assays as described herein, e.g., bySurface Plasmon Resonance (SPR) analysis using +.>2000 SPR instruments (Biacore AB, uppsala, sweden) or flow cytometry techniques.

An "immunoconjugate" is an antibody conjugated to one or more other substances, including but not limited to a cytotoxic agent or label.

The term "label" as used herein refers to a compound or composition that is directly or indirectly conjugated or fused to and facilitates detection of an agent (such as a polynucleotide probe or antibody) to which it is conjugated or fused. The label itself may be detectable (e.g., radioisotope labels or fluorescent labels) or in the case of enzymatic labels may catalyze chemical alteration of a substrate compound or composition which is detectable. The term is intended to encompass direct labeling of a probe or antibody by coupling (i.e., physically linking) a detectable substance to the probe or antibody as well as indirect labeling of the probe or antibody by reaction with another reagent that is directly labeled. Examples of indirect labeling include detection of primary antibodies using fluorescently labeled secondary antibodies and end labeling of DNA probes with biotin so that they can be detected with fluorescently labeled streptavidin.

"vector" refers to a polynucleotide capable of replication within a biological system or that can be moved between such systems. Vector polynucleotides typically contain elements such as origins of replication, polyadenylation signals, or selectable markers, which function to facilitate replication or maintenance of the polynucleotides in biological systems. Examples of such biological systems may include cells, viruses, animals, plants, and biological systems reconstituted with biological components capable of replicating vectors. The polynucleotide comprising the vector may be a DNA or RNA molecule or a hybrid of these molecules. An "expression vector" refers to a vector that can be used in a biological system or reconstituted biological system to direct translation of a polypeptide encoded by a polynucleotide sequence present in the expression vector.

An "isolated" antibody is an antibody that has been separated from components of its natural environment. In some embodiments, the antibodies are purified to greater than 95% or 99% purity, as determined by, for example, electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reverse phase HPLC). For reviews of methods for assessing antibody purity, see, e.g., flatman et al, J.chromatogrB 848:79-87 (2007).

An "isolated" nucleic acid refers to a nucleic acid molecule that has been separated from components of its natural environment. An isolated nucleic acid includes a nucleic acid molecule contained in a cell that normally contains the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or at a chromosomal location different from its natural chromosomal location.

Calculation of sequence identity between sequences was performed as follows.

To determine the percent identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps may be introduced in one or both of the first and second amino acid sequences or nucleic acid sequences for optimal alignment or non-homologous sequences may be discarded for comparison purposes). In a preferred embodiment, the length of the reference sequences aligned for comparison purposes is at least 30%, preferably at least 40%, more preferably at least 50%, 60% and even more preferably at least 70%, 80%, 90%, 100% of the length of the reference sequences. Amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position.

Sequence comparison and calculation of percent identity between two sequences can be accomplished using mathematical algorithms. In a preferred embodiment, the percentage identity between two amino acid sequences is determined using the Needlema and Wunsch ((1970) j.mol.biol.48:444-453) algorithm (available at http:// www.gcg.com) which has been integrated into the GAP program of the GCG software package, using the Blossum 62 matrix or PAM250 matrix and the GAP weights 16, 14, 12, 10, 8, 6 or 4 and the length weights 1, 2, 3, 4, 5 or 6. In yet another preferred embodiment, the percentage of identity between two nucleotide sequences is determined using the GAP program in the GCG software package (available at http:// www.gcg.com) using the NWS gapdna.CMP matrix and the GAP weights 40, 50, 60, 70 or 80 and the length weights 1, 2, 3, 4, 5 or 6. A particularly preferred set of parameters (and one that should be used unless otherwise indicated) is the Blossum 62 scoring matrix employing gap penalty 12, gap extension penalty 4, and frameshift gap penalty 5.

PAM120 weighted remainder table, gap length penalty 12, gap penalty 4) may also be used, using the e.meyers and w.miller algorithm that has been incorporated into the ALIGN program (version 2.0) ((1989) CABIOS,4: 11-17) determining the percent identity between two amino acid sequences or nucleotide sequences.

Additionally or alternatively, the nucleic acid sequences and protein sequences described herein may be further used as "query sequences" to perform searches against public databases, for example, to identify other family member sequences or related sequences.

As used herein, the term "hybridizes under low, medium, high, or very high stringency conditions" describes hybridization and washing conditions. Guidance for performing hybridization reactions can be found in Current Protocols in Molecular Biology, john Wiley & Sons, n.y. (1989), 6.3.1-6.3.6, incorporated by reference. Aqueous and non-aqueous processes are described in the references and either process may be used. Specific hybridization conditions referred to herein are as follows: 1) The low stringency hybridization conditions are at about 45 ℃ in 6X sodium chloride/sodium citrate (SSC), followed by at least two washes in 0.2X SSC,0.1% sds at 50 ℃ (for low stringency conditions the wash temperature can be increased to 55 ℃); 2) Moderate stringency hybridization conditions are one or more washes in 6 XSSC at about 45℃followed by 0.2 XSSC, 0.1% SDS at 60 ℃; 3) High stringency hybridization conditions are one or more washes in 6 XSSC at about 45℃followed by 0.2 XSSC, 0.1% SDS at 65 ℃; and preferably 4) very high stringency hybridization conditions are one or more washes in 0.5M sodium phosphate, 7% SDS at 65℃followed by 0.2 XSSC, 0.1% SDS at 65 ℃. Extremely high stringency condition (4) is a preferred condition and one condition should be used unless otherwise indicated.

The terms "host cell", "host cell line" and "host cell culture" are used interchangeably and refer to a cell into which exogenous nucleic acid is introduced, including the progeny of such a cell. Host cells include "transformants" and "transformed cells" which include the primary transformed cell and progeny derived therefrom, regardless of the number of passages. The progeny may not be exactly identical in nucleic acid content to the parent cell, but may comprise the mutation. Included herein are mutant progeny selected or selected for the same function or biological activity in the initially transformed cells. Suitable host cells for use in the present invention include prokaryotic microorganisms, such as E.coli. The host cell may also be a eukaryotic microorganism such as a filamentous fungus or yeast, or various eukaryotic cells, e.g., insect cells, etc. Vertebrate cells can also be used as hosts. For example, mammalian cell lines engineered to be suitable for suspension growth may be used. Examples of useful mammalian host cell lines include the SV40 transformed monkey kidney CV1 line (COS-7); human embryonic kidney (HEK 293 or 293F cells), 293 cells, baby Hamster Kidney (BHK), monkey kidney (CV 1), african green monkey kidney (VERO-76), human cervical cancer (HELA), canine kidney (MDCK), buffalo rat liver (BRL 3A), human lung (W138), human liver (Hep G2), chinese Hamster Ovary (CHO) cells, CHOK1SV GS-KO, CHOS, NSO, myeloma cell lines such as Y0, NS0, P3X63 and Sp2/0, etc. For reviews of mammalian host cell lines suitable for the production of proteins see, for example, yazaki and Wu, methods in Molecular Biology, volume 248 (b.K.C.Lo et al, humana Press, totowa, NJ), pages 255-268 (2003). In a preferred embodiment, the host cell is a CHO cell, e.g., a CHOs cell CHOK1SV cell or CHOK1SV GS-KO, or the host cell is a 293 cell, e.g., a HEK293 cell.

The term "agonist" or "agonistic" refers to an antibody that specifically binds to human CD40, and when bound to CD40 induces proliferation or activation of B cells and/or Dendritic Cells (DCs) or T cells. Proliferation or activation of B cells and DC and T cells can be determined by: increased B cell proliferation is assayed, or up-regulation of any of the surface markers CD23, CD80, CD83, CD86 on B cells, or CD80, CD83, CD86 and HLA-DR on DC is assayed. Agonists are capable of inducing B-cell and/or DC and/or T-cell activation in a statistically significant manner when compared to a control sample without antibody.

By "inhibit tumor cell/tumor growth" is meant a measurable decrease in tumor cell growth or tumor in vitro or in vivo upon contact with a therapeutic agent or combination of therapeutic agents, as compared to the growth of the same tumor cell or tumor in the absence of the therapeutic agent. Inhibition of tumor cells or tumor growth in vitro or in vivo can be at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 99%, or 100%.

The term "therapeutic agent" as described herein encompasses any substance that is effective in preventing or treating a disease, such as a tumor (e.g., cancer) and an infection (e.g., chronic infection), including chemotherapeutic agents, cytotoxic agents, vaccines, other antibodies, anti-infective agents, small molecule drugs, or immunomodulators.

"chemotherapeutic agents" include chemical compounds useful in the treatment of immune system disorders, including but not limited to alkylating agents; antimetabolites; anti-microtubule inhibitors, natural products; an antibiotic; an enzyme; a miscellaneous reagent; hormones and antagonists; antiestrogens; an antiandrogen; non-steroidal antiandrogens, topoisomerase inhibitors, receptor tyrosine kinase inhibitors, angiogenesis inhibitors, and the like. Exemplary chemotherapeutic agents of the invention, such as anastrozoleBicalutamide->Bleomycin sulfate->Busulfan->Busulfan injection>Capecitabine->N4-pentoxycarbonyl-5-deoxy-5-fluorocytidine, carboplatin +.>Carmustine>Chlorambucil->Cisplatin->Cladribine->Cyclophosphamide (/ -s)>Or->) Cytarabine, cytosine arabinoside +.>Cytarabine liposome injection>Dacarbazine->Dactinomycin (dactinomycin) (actinomycin D, cosmegan), daunomycin hydrochloride +.>Daunomycin citrate liposome injection>Dexamethasone, docetaxel +.>Doxorubicin hydrochloride Etoposide->Fludarabine phosphate->5-fluorouracilFluotamide->tezacitibine, gemcitabine (difluoro deoxycytidine), hydroxyureaIdarubicin->Ifosfamide->Irinotecan- >L-asparaginase->Calcium folinate, melphalan->6-mercaptopurine->Methotrexate>Mitoxantrone (mitoxantrone), milostat (mylotarg), paclitaxel +.>Phoenix (yttrium 90/MX-DTPA), penstatin and polifeprosan 20 in combination with carmustine implant +.>Tamoxifen citrate->Teniposide6-thioguanine, thiotepa, tirapazamine>Topotecan hydrochloride for injection>Vinblastine->Vincristine->Vinorelbine (vinorelbine), ibrutinib, gilidellinib (idelalisib) and belantomab-vildagliptin (brentuximab vedotin), and pharmaceutically acceptable ones of any of the foregoingSalts, acids or derivatives. . The definition also includes anti-hormonal drugs such as antiestrogens for modulating or inhibiting the action of hormones on tumors, including, for example, tamoxifen, raloxifene, aromatase inhibiting 4 (5) -imidazole, 4-hydroxy tamoxifen, trowoxifene, keoxifene, LY117018, onapristone and toremifene, and anti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide acetate and goserelin; and pharmaceutically acceptable salts, acids or derivatives of any of the above.

The term "cytotoxic agent" is used herein to refer to a substance that inhibits or prevents cellular function and/or causes cell death or destruction. Exemplary cytotoxic agents include, but are not limited to: radioisotopes, e.g. iodine @, of ¹³¹ I、 ¹²⁵ I、 ¹²³ I and ¹²¹ i) The carbon is ¹⁴ C) Sulfur ³⁵ S, tritium ³ H) The indium is ¹¹⁵ In、 ¹¹³ In、 ¹¹² In and ¹¹¹ in, technetium ] ⁹⁹ Tc), thallium ²⁰¹ Ti, ga ] ⁶⁸ Ga、 ⁶⁷ Ga and Pd% ¹⁰³ Pd and molybdenum% ⁹⁹ Mo and xenon ¹³³ Xe and F ¹⁸ F)、 ¹⁵³ Sm、 ¹⁷⁷ Lu、 ¹⁵⁹ Gd、 ¹⁴⁹ Pm、 ¹⁴⁰ La、 ¹⁷⁵ Yb、 ¹⁶⁶ Ho、 ⁹⁰ Y、47Sc、 ¹⁸⁶ Re、 ¹⁸⁸ Re、 ¹⁴² Pr、 ¹⁰⁵ Rh、 ⁹⁷ Ru、 ⁶⁸ Ge、 ⁵⁷ Co、 ⁶⁵ Zn、 ⁸⁵ Sr、 ³² P、 ¹⁵³ Gd、 ¹⁶⁹ Yb、 ⁵¹ Cr、 ⁵⁴ Mn、 ⁷⁵ Se、 ¹¹³ Sn and Sn ¹¹⁷ Tin; a growth inhibitor; alkylating agents (including but not limited to nitrogen mustard, ethyleneimine derivatives, alkyl sulfonates, nitrosoureas, and triazenes), e.g., uracil nitrogen mustard, nitrogen mustard (Chlormethine), cyclophosphamide (CYTOXAN) ^TM ) Ifosfamide (fosfamide), melphalan, chlorambucil, guanadine, triethylenemelamine, triethylenethiophosphamine (triethylenethiophosphamine), busulfan, carmustine, lomustine, streptozotocin, dacarbazine and temozolomide; or antimetabolites (including but not limited toIn folic acid antagonists, pyrimidine analogs, purine analogs, and adenosine deaminase inhibitors): methotrexate, 5-fluorouracil, fluorouridine, cytarabine, 6-mercaptopurine, 6-thioguanine, fludarabine phosphate, pentostatin, and gemcitabine. In some embodiments, exemplary cytotoxic agents of the invention include anti-microtubule agents, topoisomerase inhibitors, antimetabolites, mitotic inhibitors, alkylating agents, anthracyclines, vinca alkaloids, intercalators, active agents capable of interfering with signal transduction pathways, pro-apoptotic active agents, proteasome inhibitors, and irradiation (e.g., local or systemic irradiation (e.g., gamma irradiation).

The term "small molecule drug" refers to a low molecular weight organic compound capable of modulating biological processes. A "small molecule" is defined as a molecule having a molecular weight of less than 10kD, typically less than 2kD and preferably less than 1 kD. Small molecules include, but are not limited to, inorganic molecules, organic molecules containing inorganic components, molecules containing radioactive atoms, synthetic molecules, peptidomimetics, and antibody mimetics. As a therapeutic agent, small molecules may be more cell permeable, less susceptible to degradation, and less prone to eliciting an immune response than large molecules. For descriptions of small molecules, such as peptide mimics of antibodies and cytokines, and small molecule toxins see, e.g., casset et al, (2003) biochem. Biophysis. Res. Commun.307:198-205; muyldermans (2001) J.Biotechnol.74:277-302; li (2000) nat. Biotechnol.18:1251-1256; apostolopoulos et al, (2002) curr.med.chem.9:411-420; monfardini et al, (2002) Curr.Pharm. Des.8:2185-2199; dominages et al, (1999) Nat. Structure. Biol.6:652-656; sato and Sone (2003) biochem.j.371:603-608; U.S. patent No. 6,326,482.

The term "anti-infective agent" includes any molecule that specifically inhibits or eliminates the growth of microorganisms, such as viruses, bacteria, fungi, or protozoa, e.g., parasites, but is not fatal to the host at the concentration and dosing interval of administration. As used herein, the term anti-infective active agent includes antibiotics, antibacterial agents, antiviral agents, antifungal agents, and antiprotozoal agents. In a particular aspect, the anti-infective agent is non-toxic to the host at the concentration and interval of administration.

Antibacterial anti-infective agents or antibacterial agents can be broadly classified as bactericidal (i.e., direct kill) or bacteriostatic (i.e., prevent division). Antibacterial anti-infective agents can be further sub-classified as either narrow spectrum antibacterial agents (i.e., affecting only small bacterial subtypes, e.g., gram negative, etc.) or broad spectrum antibacterial agents (i.e., affecting a broad class). Examples include amikacin, gentamicin, geldanamycin, herbimycin, mupirocin, nitrofurantoin, pyrazinamide, quinioprene/dalfopristin, rifampin/isononamide, or tinidazole, among others.

The term "antiviral agent" includes any agent that inhibits or eliminates viral growth, disease, and/or survival. This includes, for example, acyclovir, cidofovir, zidovudine, didanosine (ddI, VIDEX), zalcitabine (ddC, HIVID), stavudine (d 4T, zero), lamivudine (3 tc, epivir), abacavir (ziage), emtricitabine (EMTRIVA), and the like.

The term "antifungal agent" includes any agent that inhibits or eliminates fungal growth, disease causing and/or survival. This includes, for example, natamycin, spinosad, filipin, nystatin, amphotericin B, candesamin, patchouli (patchouli), neem seed Oil (neem seed Oil), coconut Oil (Coconut Oil) and the like.

The term "antiprotozoal agent" includes any substance that inhibits or eliminates the growth, onset and/or survival of a protozoan organism (e.g., parasite). Examples of the antimalarial agent include antimalarial agents such as quinine, quinidine, and the like.

The term "immunomodulator" as used herein refers to a natural or synthetic active agent or drug that inhibits or modulates an immune response. The immune response may be a humoral response or a cellular response. Immunomodulators comprise immunosuppressants.

The term "immune checkpoint molecule" means a class of inhibitory signal molecules present in the immune system that avoid tissue damage by modulating the persistence and intensity of immune responses in peripheral tissues and are involved in maintaining tolerance to autoantigens (Pardoll DM., the blockade of immune checkpoints in Cancer immunology. Nat Rev Cancer,2012, 12 (4): 252-264). It was found that one of the reasons that tumor cells can evade the immune system in vivo and proliferate uncontrollably is to use the inhibitory signaling pathway of immune checkpoint molecules, thereby inhibiting T lymphocyte activity, so that T lymphocytes cannot effectively exert a killing effect on tumors (Yao S, zhu Y and Chen l., advances in targeting cell surface signaling molecules for immune modulation. Nat Rev Drug discovery, 2013, 12 (2): 130-146). Immune checkpoint molecules include, but are not limited to, programmed death 1 (PD-1), PD-L1, PD-L2, cytotoxic T lymphocyte antigen 4 (CTLA-4), LAG-3, and TIM-3.

The term "costimulatory molecule" refers to a corresponding binding partner on a T cell that specifically binds to a costimulatory ligand, thereby mediating a costimulatory response (such as, but not limited to, proliferation) of the T cell. Costimulatory molecules are cell surface molecules other than antigen receptors or their ligands that contribute to an effective immune response. Costimulatory molecules include, but are not limited to, MHC class I molecules, TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocyte activation molecules (SLAM proteins), activation of NK cell receptors, OX40, CD40, GITR, 4-1BB (i.e., CD 137), CD27, and CD28. In some embodiments, the "costimulatory molecule" is OX40, GITR, 4-1BB (i.e., CD 137), CD27, and/or CD28.

The term "cytokine" is a generic term for proteins released by one cell population that act as intercellular mediators on another cell. Examples of such cytokines are lymphokines, monokines, interleukins (IL), such as IL-1, IL-1. Alpha., IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-11, IL-12, IL-15; tumor necrosis factors such as TNF- α or TNF- β; and other polypeptide factors including LIF and Kit Ligand (KL) and gamma interferon. As used herein, the term cytokine includes proteins from natural sources or from recombinant cell cultures and biologically active equivalents of native sequence cytokines, including small molecule entities produced by artificial synthesis, and pharmaceutically acceptable derivatives and salts thereof.

The term "inhibitor" or "antagonist" includes substances that reduce certain parameters (e.g., activity) of a given molecule (e.g., an immune checkpoint molecule). For example, this term includes substances that cause a given molecule to be inhibited by at least 5%, 10%, 20%, 30%, 40% or more of activity (e.g., PD-L1 activity). Thus, inhibition need not be 100%.

The term "activator" includes substances that increase certain parameters (e.g., activity) of a given molecule (e.g., a co-stimulatory molecule). For example, this term includes substances that cause a given molecule to be increased by at least 5%, 10%, 20%, 30%, 40% or more in activity (e.g., OX40 activity). Thus, the activation need not be 100%.

The term "pharmaceutical adjuvant" refers to diluents, adjuvants (e.g., freund's adjuvant (complete and incomplete)), excipients, carriers or stabilizers, etc. for administration with the active substance.

The term "pharmaceutical composition" refers to a composition that exists in a form that is effective to allow the biological activity of the active ingredient contained therein, and that does not contain additional ingredients that have unacceptable toxicity to the subject to whom the composition is administered.

The term "combination" refers to a kit of parts for combined administration or a fixed or non-fixed combination in dosage unit form, wherein two or more therapeutic agents may be administered independently at the same time or separately at intervals, particularly where these intervals allow the combination partners to demonstrate a co-operation, e.g. a co-effect. The term "fixed combination" refers to the simultaneous administration of an antibody of the invention and a combination partner (e.g., other therapeutic agent, such as an immunomodulatory agent, e.g., an immunosuppressant or anti-inflammatory agent) to a patient in the form of a single entity or dose. The term "non-fixed combination" means that the antibodies of the invention and a combination partner (e.g., other therapeutic agent, such as an immunomodulatory agent, such as an immunosuppressant or anti-inflammatory agent) are administered to a patient as separate entities, simultaneously, concurrently or sequentially, without specific time constraints, wherein such administration provides therapeutically effective levels of both compounds in the patient. The latter is also applicable to cocktail therapies, such as administration of three or more therapeutic agents. In a preferred embodiment, the pharmaceutical combination is a non-fixed combination.

"immunogenicity" refers to the ability of a particular substance to elicit an immune response. Tumors are immunogenic and enhancing tumor immunogenicity aids in the elimination of tumor cells by an immune response.

An "immune response" refers to a biological response in a vertebrate against a foreign substance that protects the organism from these substances and diseases caused thereby. The immune response is mediated by the action of cells of the immune system (e.g., T lymphocytes, B lymphocytes, natural Killer (NK) cells, macrophages, eosinophils, mast cells, dendritic cells, or neutrophils) and soluble macromolecules produced by these cells or the liver, including antibodies, cytokines, and complement, which result in selective targeting, binding, injury, destruction, and/or elimination of invading pathogens, cells or tissues infected with pathogens, cancerous or other abnormal cells, or (in the case of autoimmune or pathological inflammation) normal human cells or tissues from the vertebrate organism. Immune responses include, for example, activation or suppression of T cells, e.g., effector T cells or Th cells, such as cd4+ or cd8+ T cells, or suppression or depletion of Treg cells. "T effect" ("T) _eff ") cells refer to T cells (e.g., cd4+ and cd8+ T cells) with cytolytic activity, and T helper (Th) cells that secrete cytokines and activate and direct other immune cells, but do not include regulatory T cells (Treg cells).

The term "effective amount" refers to an amount or dose of an antibody or fragment or conjugate or composition of the invention that, upon administration to a patient in single or multiple doses, produces a desired effect in a patient in need of treatment or prevention. The effective amount can be readily determined by the attending physician as a person skilled in the art by considering a number of factors: species such as mammals; its size, age and general health; specific diseases involved; the extent or severity of the disease; response of individual patients; specific antibodies administered; mode of administration; the bioavailability characteristics of the administration formulation; a selected dosing regimen; and the use of any concomitant therapy.

"therapeutically effective amount" means an amount effective to achieve the desired therapeutic result at the desired dosage and for the desired period of time. The therapeutically effective amount of the antibody or antibody fragment or conjugate or composition thereof may vary depending on a variety of factors such as the disease state, age, sex and weight of the individual, and the ability of the antibody or antibody portion to elicit a desired response in the individual. A therapeutically effective amount is also an amount in which any toxic or detrimental effect of the antibody or antibody fragment or conjugate or composition thereof is less than a therapeutically beneficial effect. The "therapeutically effective amount" preferably inhibits a measurable parameter (e.g., swelling rate, etc.) by at least about 20%, more preferably by at least about 40%, even more preferably by at least about 50%, 60% or 70% and still more preferably by at least about 80% or 90% relative to an untreated subject. The ability of a compound to inhibit a measurable parameter (e.g., swelling rate) can be evaluated in an animal model system that predicts efficacy in human autoimmune disease or inflammation.

"prophylactically effective amount" means an amount effective to achieve the desired prophylactic result at the desired dosage and for the desired period of time. Typically, since the prophylactic dose is administered in the subject prior to or at an earlier stage of the disease, the prophylactically effective amount will be less than the therapeutically effective amount.

As used herein, "individual" or "subject" may be used interchangeably, including mammals. Mammals include, but are not limited to, domesticated animals (e.g., cattle, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In some embodiments, the individual or subject may be a mammal, e.g., a primate, preferably a higher primate, e.g., a human (e.g., a patient suffering from or at risk of suffering from a disease described herein). In one embodiment, the subject has or is at risk of having a disease described herein (e.g., a tumor or infection as described herein). In certain embodiments, the subject receives or has received other treatments, such as chemotherapy and/or radiation therapy. Alternatively or in combination, the subject is immunocompromised by infection or is at risk of being immunocompromised by infection.

The term "combination therapy" refers to the administration of two or more therapeutic agents or modes of treatment (e.g., radiation therapy or surgery) to treat IL-23-related disorders as described in the present disclosure. Such administration includes co-administration of the therapeutic agents in a substantially simultaneous manner, e.g., in a single capsule with a fixed ratio of active ingredients. Alternatively, such administration includes co-administration of the individual active ingredients in multiple or separate containers (e.g., tablets, capsules, powders, and liquids). The powder and/or liquid may be reconstituted or diluted to the desired dosage prior to administration. In addition, such administration also includes the use of each type of therapeutic agent in a sequential manner at about the same time or at different times. In either case, the treatment regimen will provide a beneficial effect of the pharmaceutical combination in treating the disorders or conditions described herein.

As used herein, "treating" refers to slowing, interrupting, blocking, alleviating, stopping, reducing, or reversing the progression or severity of an existing symptom, disorder, condition, or disease.

As used herein, "preventing" includes inhibition of the occurrence or progression of a disease or disorder or a symptom of a particular disease or disorder. In some embodiments, subjects with a family history of immune system disease (autoimmune disease or inflammation) are candidates for prophylactic regimens. Generally, in the context of an immune system disorder (autoimmune disease or inflammation), the term "preventing" refers to administration of a drug prior to the occurrence of a sign or symptom of an immune system disorder (autoimmune disease or inflammation), particularly prior to the occurrence in a subject at risk of an immune system disorder (autoimmune disease or inflammation).

"subject/patient sample" refers to a collection of cells or fluids obtained from a patient or subject. The source of the tissue or cell sample may be solid tissue, like an organ or tissue sample or biopsy or puncture sample from fresh, frozen and/or preserved; blood or any blood component; body fluids such as cerebrospinal fluid, amniotic fluid (amniotic fluid), peritoneal fluid (ascites), or interstitial fluid; cells from any time of gestation or development of a subject. Tissue samples may contain compounds that are not naturally intermixed with the tissue in nature, such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics, and the like.

These and other aspects and embodiments of the invention are described in and exemplified by the following examples in the figures (which are briefly described to follow) and in the following detailed description. Any or all of the features discussed above and throughout this application may be combined in various embodiments of the invention. The following examples further illustrate the invention, however, it is to be understood that the examples are presented by way of illustration and not limitation, and that various modifications may be made by those skilled in the art.

Examples

EXAMPLE 1 construction of reporter cell line Jurkat/NF-. Kappa.B-GFP+hCD 40

NF-. Kappa.B-GFP reporter Gene the promoter of the lentiviral system (QIAGEN, CCS-013G) has multiple NF-. Kappa.B transcription factor binding elements, and activation of this promoter drives GFP expression. Jurkat cells (ATCC, TIB-152) were infected with NF- κB-GFP reporter lentivirus and the lentivirus-infected Jurkat cells were screened with puromycin (InvivoGen, ant-pr-1) according to the instructions of the kit. The cells screened with puromycin were stimulated by addition of 10ng/ml TNF alpha protein (Sino Biological, 10602-HNAE) and a 24h post flow cytometer (BD, FACSAria III) sorted for a subset of GFP fluorescent cells to obtain Jurkat/NF- κB-GFP cells.

Human CD40 CDS (Sino Biological, HG 10774-M) was constructed into lentiviral core plasmid pCDH (System Biosciences), the obtained lentiviral core plasmid and helper plasmid pPACKH1-GAG, pPACKH1-REV, pVSVG (both from System Biosciences) were co-transfected with PEI (polyscience, 24885-2) into 293FT cells (Thermo Fisher Scientific, R70007) at 1:1:1 using standard procedure, the supernatant containing human CD40 lentivirus was collected by changing the solution after 6h transfection, culturing at 37℃in DMEM medium (Lifetechnologies, C11995500 CP) containing 10% fetal bovine serum (Biological Industries, 04-001-1A) and further culturing for 48 h.

The positive control CD40L sequence used herein is from patent WO 2016/177771 (SEQ)ID NO:15 The negative control HEL sequence is shown in table 10. The DNA sequences of CD40L and HEL were synthesized by the Jin Weizhi company. For CD40L and HEL purification, DNA sequences were inserted into eukaryotic expression vector pFUSE (invitofgen, pFUSE-hg1fc 1) and transfected with PEI into 293F cells using standard procedures (Thermo Fisher Scientific, R79007); cells were shake cultured at 37℃using Freestyle medium (Lifetechnologies, 12338026). Supernatants were collected after 7 days of culture and Superdex was used on AKTA System (GE) ^TM 200 The Increase pre-packed column (GE, 28-9909-44) was purified.

CD40 signaling can activate NF- κB pathway, and CD40L stimulation will induce GFP expression if CD40 is successfully expressed on the surface of Jurkat/NF- κB-GFP cell membrane. The constructed Jurkat/NF- κB-GFP cells obtained by sorting as described above were infected with the human CD40 lentivirus supernatant obtained as described above, and after 16h, 100nM of the obtained CD40L protein (as positive control) and HEL (as negative control) were added for stimulation, and after 24h the flow cytometer single cell sorted the cells with the strongest GFP fluorescence into 96 well plates.

Cells in the 96-well plates were cultured in RPMI1640 medium (Life technologies, C11875500 CP) containing 10% fetal bovine serum (Biological Industries, 04-001-1A) for 2 weeks, 100nM of the trimerized CD40L protein (as positive control) and HEL (as negative control) were added after single cells grew, and a strong positive, HEL-negative monoclonal CD40L was selected as Jurkat/NF-. Kappa.B-GFP+hCD 40 (NF-. Kappa.3-GFP+hCD 40 for short) cells for subsequent experiments. As a result, as shown in FIG. 1, the Jurkat/NF-. Kappa.B-GFP+hCD 40 monoclonal GFP positive rate after CD40L stimulation was 94.7%, and HEL stimulation was not activated.

Example 2 screening of phage display antibody libraries for CD40 binding antibodies

30 healthy adult peripheral blood was purchased from Tianjin blood center and Shanghai second general biological company, and PBMC was obtained by centrifugation using ficoll separating liquid (Denyang, LDS 1075), centrifugation conditions: 20 ℃,2000rpm, 5-step down 0 mode for 30 minutes. Human natural antibody libraries were constructed using PBMCs obtained as described above, the construction of the libraries being conventional (phage display, tim Clackson and Henry b. Random combinations of antibody heavy and light chain variable regions will be obtainedThe single-chain antibody scFv is displayed at the N end of phage capsid protein pIII to obtain phage display antibody library with the library capacity of 10 ¹⁰ 。

Phage selection is a conventional technique (Phage Display: A Laboratory Manual, carlos F Barbas III). First, biotinylated CD40 protein (acrobiosystems, TN5-H82F 9) was incubated with phage display antibody libraries obtained as described above for 2H at room temperature. After the incubation, 150ul of streptavidin beads Dynabeads M280 (Life technologies, 11205D) were added directly and incubated on a room temperature mixer for 30min. Phage not bound to antigen were washed off with 0.05% PBS-tween (PBS: lifetechnologies,70011044; tween: sigma-Aldrich, 9005-64-5), and finally phage bound to antigen was eluted with 0.2M glycine-HCl (pH 2.2). Coli XL1-blue (Agilent, 200236) was infected with the eluted phage, and amplified by adding helper phage VCSM13 (Agilent, 200251) for the next round of screening. Three rounds of screening were performed in total. After three rounds, antibody phages binding to CD40 were enriched and the screening results are shown in table 1.

TABLE 1 screening of CD40 binding antibodies from human Natural antibody libraries Using phage display technology

To initially evaluate the positive rate of screening for CD40 binding antibodies, we picked the second and third rounds of 29 phage monoclonal, respectively, for phage ELISA analysis. Phage ELISA is a conventional technique, specifically as follows: and (3) selecting a second phage monoclonal and a third phage monoclonal from phage monoclonal cultured in the deep-hole plate, shaking at 37 ℃ and 300rpm until OD=0.5-0.8, adding a helper phage VCSM13, amplifying, and inducing phage at 30 ℃ overnight for antibody expression. 1. Mu.g/ml of human CD40 (Acrobiosystems, CD 0-H5228) was coated on ELISA plates (Corning, 3690) and incubated overnight at 4 ℃. The overnight induced phage supernatant was added to the ELISA plate after overnight incubation, incubated for 1h at room temperature, and washed 8 times with 0.05% PBST. BSA (Solarbio, A8020-100) was used as a negative control. Bound antibody phages were detected by addition of HRP-conjugated anti-M13 (GE, 27-9421-01, M13 being phage capsid protein). More than 3-fold higher CD40 binding signal than BSA control was defined as positive clones. The results are shown in FIG. 2, wherein the positive rates of the second and third rounds of screening were 6.9% and 27.6%, respectively.

Example 3 agonist antibody screening of Co-stimulatory molecule CD40

First, we subcloned the antibodies enriched for the third round of phage display into the secreted lentiviral vector pCDH, while cloning N27 ScFv into the secreted lentiviral vector pCDH as a negative control, the N27 ScFv sequences are shown in table 11. Co-transfecting lentiviral core plasmid and helper plasmid pPACKH1-GAG, pPACKH1-REV and pVSVG into 293FT cells according to the ratio of 1:1:1, transferring after 6h of transfection, culturing in DMEM medium containing 10% fetal bovine serum (Biological Industries, 04-001-1A) at 37 ℃ for 48h, and collecting supernatant containing CD40 binding antibody lentiviral library; HEK293 cells were infected with 50000pg of the obtained lentiviral library of CD40 binding antibodies, after 16h of infection, the lentiviral-containing medium was removed by centrifugation, fresh medium was added, the infected HEK293 cells were sorted into 96-well plates using flow sorting technique, so that each well contained only 1 infected HEK293 cell, the cells were cultured for 3 weeks, and the supernatant was taken after the cells reached a certain number. Supernatant addition Jurkat/NF-. Kappa.B-GFP+hCD 40 reporter cells 3X 10 obtained as described above ⁵ Simultaneously adding 2.5 mug/ml goat anti-human Fc (SouthernBiotech, SBA-2048-01) secondary antibody, and crosslinking the antibody secreted by the cells to enhance the activation strength of the agonist antibody; after 24h of culture, the activity of the cell supernatant was tested, and the detection result of the positive antibody is shown in FIG. 3; extracting antibody genes from HEK293 cells with positive cell supernatant activity, constructing pFUSE vectors, and sequencing to obtain positive antibody VH and VL sequences, wherein the obtained two positive antibodies are NK003 and NK004, and the sequences (including CDR, VH/VL, heavy chain and light chain) are shown in the sequences in tables 4 to 8 of the sequence table.

EXAMPLE 4 expression and purification of full-length antibody IgG

The expression and purification of the antibody are conventional methods, and are specifically as follows:

synthesizing screened agonist antibody NK003 and NK004 heavy chains according to antibody sequencesAnd light chain DNA (Jin Weizhi), cloned separately into vector pFUSE, and both plasmids containing heavy and light chains, respectively, were transiently co-transfected 1:1 into 293F suspension cells with PEI using standard procedures to express full length antibodies. Superdex was used on AKTA System after 1 week of culture ^TM 200 IncreatePre-packed column purification.

SDS-PAGE was used to identify purified antibodies and the aggregation of antibodies was analyzed using a size exclusion pre-cartridge Superdex 200. The results are shown in fig. 4, where NK003 elution peak shape was symmetric and retention time at elution was consistent with that of single molecular weight monoclonal antibody, while small amount of aggregates were present in the antibody.

Example 5 in vitro characterization of human CD40 antibody NK003, NK004

5.1 Binding of NK003, NK004 to CD40

The binding selectivity of NK003 was evaluated by direct ELISA with TNFR family members OX40, 4-1BB and GITR. PBS solution containing 1. Mu.g/ml of human-derived OX40 (Acrobiosystems, OX 0-H5224), 4-1BB (Acrobiosystems, 41B-H5227), GITR (Acrobiosystems, GIR-H5228) and CD40 (Acrobiosystems, CD 0-H5228) was coated on ELISA plates and incubated overnight at 4 ℃. NK003 diluted to different concentrations with PBS as obtained in example 4 was added: 0.625. Mu.g/ml, 1.25. Mu.g/ml, 2.5. Mu.g/ml and 5. Mu.g/ml, after incubation for 30min at room temperature, washed 8 times with 0.05% PBS-Tween. Bound NK003 was detected by addition of goat anti-human HRP conjugated Fc antibody (Southem Biotech, 2048-05). As shown in fig. 5A, NK003 selectively bound to human CD40 with or without low binding to other TNFR family proteins tested.

To evaluate NK004 binding to CD40, 1. Mu.g/ml of a PB solution of human CD40 (Acrobiosystems, CD 0-H5228) was coated on ELISA plates and incubated overnight at 4 ℃. PBS obtained as in example 4 was added to dilute to different concentrations of NK004:0.002nM, 0.016nM, 0.13nM, 1nM, 8nM, 64nM and 500nM, HEL as negative control antibody, after 30min incubation at room temperature, 8 washes with 0.05% PBS-Tween. Bound NK004 was detected by addition of goat anti-human HRP conjugated Fc antibody. As shown in fig. 5B, NK004 may bind to human CD 40.

5.2 NK003 blocks CD40L binding to CD40

The effect of NK003 on CD40L binding to CD40 was evaluated by ELISA. First, CD40L was biotinylated according to the instructions using the protein biotinylation kit (GeneCopoeia, BI 001). The ELISA plates were coated with 1. Mu.g/ml human CD40 and incubated overnight at 4 ℃. PBS obtained as in example 4 was added to dilute to different concentrations of NK003: 0.156. Mu.g/ml, 0.313. Mu.g/ml, 0.625. Mu.g/ml, 1.25. Mu.g/ml, 2.5. Mu.g/ml, 5. Mu.g/ml, 10. Mu.g/ml and 20. Mu.g/ml HEL as control antibody, after incubation for 1h at room temperature, 2.5. Mu.g/ml biotinylated CD40L was added and after incubation for 30min at room temperature, 0.05% PBS-Tween was washed 8 times. The OD405 value was read by adding Strepitavidin-HRP using a microplate reader (SpectraMax i3 x) and the amount of binding of CD40L to CD40 was measured. The results are shown in FIG. 6, where NK003 blocks the binding of CD40 to CD40L.

5.3 NK003, NK004 activate NF-. Kappa.B-GFP+hCD 40 reporter cell lines in cross-linked form

In this example, the activation of the NF-. Kappa.B-GFP+hCD 40 reporter cell line by the NK003, NK004 antibodies was detected by flow cytometry and the EC50 of both antibodies was determined as follows:

respectively taking 3×10 ⁵ NF- κb-gfp+hcd40 reporter cells were obtained as in example 1 and NK003 or NK004 diluted to different concentrations as obtained in example 4 were added respectively: 0.001. Mu.g/ml, 0.005. Mu.g/ml, 0.01. Mu.g/ml, 0.05. Mu.g/ml, 0.1. Mu.g/ml, 0.5. Mu.g/ml, 1. Mu.g/ml, 5. Mu.g/ml and 10. Mu.g/ml, HEL antibody was used as a negative control; for cross-linking, the secondary antibodies, goat anti-human Fc, were added simultaneously at a concentration of 2.5. Mu.g/ml for each group. After co-incubation in RPMI 1640 medium (Lifetechnologies, C11875500 CP) containing 10% fetal bovine serum (Biological Industries, 04-001-1A) at 37℃for 24h, PBS was washed three times and analyzed using a flow cytometer. The obtained data were fitted to a curve using GraphPad Prism 6.0 and EC50 was calculated. Results as shown in fig. 7, activation of CD40 by nk003 was dependent on secondary anti-crosslinking, activating NF- κb-gfp+hcd40 reporter cell line in crosslinked form with an EC50 of 2nM (fig. 7A). Activation of CD40 by NK004 was independent of secondary antibody cross-linking, activating NF-. Kappa.B-GFP+hCD 40 reporter cell lines in constitutive mode with an EC50 of 4Nm when secondary antibody was added (FIG. 7B).

5.4 quantitative analysis of NK003 kinetics and affinity by surface plasmon resonance

BiacoreT200 (GE Healthcare) was used to detect the affinity of antibodies NK003, NK 004. NK003, NK004 were flowed through the Protein A chip at 10. Mu.L/min and captured on the chip. The antigen human CD40 recombinant protein to be tested (Acrobiosystems, CD 0-H5228) was diluted with a gradient of Running buffer (HBS-EP+, GE) at a concentration of 2. Mu.M, 1. Mu.M, 500nM, 250nM, 125nM, 62.5nM, 31.25nM. The different concentrations of CD40 were passed through the antibody-capturing chip at a flow rate of 30. Mu.L/in, and bound for 120s, and then the chip was passed through a Running buffer at a flow rate of 30. Mu.L/min for 240s, whereby the antigen was gradually dissociated from the antibody-capturing chip. Data processing was performed using Biacore T200 instrument suite software BIAevaluation software S, calculating the binding constant (Ka), dissociation constant (Kd) and balancing dissociation constant (K) _D ). The results are shown in Table 2.

Table 2 affinity of antibodies to human CD40 using surface plasmon resonance technology platform

Sensor for detecting a position of a body	Antibodies to	Antigens	ka(1/Ms)	kd(1/s)	K _D (M)
						Protein A	NK003	CD40	3.67E+04	1.21E-02	3.31E-07
Protein A	NK004	CD40	8.87E+04	3.90E-02	4.40E-07

5.5 NK003 Cross-reaction

The binding of NK003 to 293FT cells expressing rhesus and human CD40 was detected by flow cytometry and the cross-reactivity of NK003 with rhesus and human CD40 was determined.

CDS regions (Jin Weizhi synthesis) of rhesus CD40 (NCBI, NM-001265862.1) and human CD40 (NCBI, NM-001250.5) were cloned into pCDH vectors, transiently transfected into 293FT cells with PEI using standard procedures, plated for 6h, incubated in DMEM medium with 10% fetal bovine serum (Biological Industries, 04-001-1A), and after 48h expression obtained cells expressing rhesus CD40 and cells expressing human CD40, respectively.

Respectively taking 3×10 ⁵ 293FT cells expressing rhesus and human CD40 obtained as described above were added with NK003 diluted with PBS to different concentrations prepared as described in example 4: 0.001. Mu.g/ml, 0.005. Mu.g/ml, 0.01. Mu.g/ml, 0.05. Mu.g/ml, 0.1. Mu.g/ml, 0.5. Mu.g/ml, 1. Mu.g/ml, 5. Mu.g/ml and 10. Mu.g/ml HEL 10. Mu.g/ml were used as negative controls, cells were incubated with different concentrations of NK0034℃for 30min, washed three times with PBS, alexa Fluro 488 goat anti-human Fc fluorescent secondary antibody (Lifetechnologies, A11013) was added at 1:100 and incubated at 4℃for 30min in the dark and analyzed by flow cytometry. The graph pad Prism 6.0 was used to fit the curve and calculate the EC50, and as shown in fig. 8, NK003 was found to cross-react with both rhesus CD40 (fig. 8B) and human CD40 (fig. 8A), with an EC50 of 8nM for rhesus CD40 and 10nM for human CD 40.

5.6 NK003 induces apoptosis in tumor cells

Lymphoma cells Raji and Ramos membrane surface have CD40 expression, we used Raii cells (ATCC, CRL-7936) and Ramos cells (ATCC, CRL-1596) to examine the ability of NK003 to promote apoptosis of tumor cells.

Raji and Ramos cells were grown at 3X 10 ⁵ Density of/mL was inoculated in 24-well plates, cultured in RPMI 1640 medium containing 10% fetal bovine serum (Lifetechnologies, 10091148), cell inoculation with simultaneous addition of PBS diluted to different concentrations of NK003 prepared as described in example 4: 0.03. Mu.g/ml, 0.06. Mu.g/ml, 0.09. Mu.g/ml, 0.3. Mu.g/ml, 0.6. Mu.g/ml, 0.9. Mu.g/ml, 3. Mu.g/ml, 6. Mu.g/ml and 9. Mu.g/ml, HEL 9. Mu.g/ml were used as negative controls, while goat anti-human Fc was added as a cross-linker at a concentration of 2.5. Mu.g/ml per well. After 24h of co-incubation, PBS was washed three times, PE-CD95 (Biolegend, 305611) was added 1:100 and incubated for 30min at 4℃in the absence of light. The apoptosis condition is detected by analyzing the expression (expressed as MFI) of the apoptosis marker molecule CD95 by using a flow cytometer. Curves were fitted using GraphPad Prism 6.0 and EC50 was calculated. As a result, as shown in fig. 9, nk003 promoted apoptosis of Raji with EC50 of 2.6nM (fig. 9A); NK003 promoted apoptosis of Ramos cells with an EC50 of 3nM (FIG. 9B).

EXAMPLE 6 Primary T cell Activity assay

6.1 DC cell activation

Human Peripheral Blood Mononuclear Cells (PBMC) were used to examine the ability of NK003 to activate Dendritic Cells (DCs). The isolation of DC cells is a conventional method, and is specifically as follows: PBMC (Hemacare, PB 009C-3) were cultured in RPMI1640 medium containing 10% foetal calf serum (Lifetechnologies, 10091148) at 37℃and adherent monocytes were harvested after 6 hours; the supernatant was removed, and a serum-free RPMI1640 medium containing 100ng/mL GM-CSF (R & D Systems, 204-IL) and 10ng/mL IL4 (R & D Systems, 215-GM) was added for culture at 37 ℃; three days later, half of the medium was replaced, and GM-CSF and IL4 were supplemented to 100ng/mL and 10ng/mL; the suspension cells were replaced with a new flask on the sixth day of culture, and the culture was continued for 24 hours using the same serum-free RPMI1640 medium containing 100ng/mL GM-CSF and 10ng/mL IL4 to obtain DC cells.

On the seventh day, the DC cells obtained by the induction were cultured at 1X 10 ⁵ Number of individual wells/well was inoculated in 96 wellsPlates, add different concentrations of NK003: 0.01. Mu.g/ml, 0.05. Mu.g/ml, 0.1. Mu.g/ml, 0.5. Mu.g/ml, 1. Mu.g/ml, 10. Mu.g/ml and 100. Mu.g/ml HEL as negative controls; for cross-linking, goat anti-human Fc was added simultaneously to each group at a concentration of 2.5. Mu.g/ml.

After incubation of the above DC cells with antibodies for 24h, PBS was washed three times, PE-CD11c (DC cell marker molecule, biolegend, 117309) and APC-CD86 (DC cell activation marker molecule, biolegend, 105007) were added 1:100 and incubated at 4℃for 30min in the absence of light. DC cell activation was measured using a flow cytometer and expressed as the MFI of APC-CD 86. Activation of DC cells can result in a stronger anti-tumor T cell response. Curves were fitted using GraphPad Prism 6.0 and EC50 was calculated. As a result, as shown in FIG. 10, NK003 significantly activated human DC cells with an EC50 of 2nM (FIG. 10A) and an EC50 of 8nM after addition of a crosslinking agent (goat anti-human Fc, anti-Fc) (FIG. 10B)And the intensity of NK003 activated DC in crosslinked form was significantly enhanced (MFI significantly higher) than that without crosslinking.

6.2 proliferation of B cells

Human Peripheral Blood Mononuclear Cells (PBMC) were used to examine the ability of NK003 to induce B cell proliferation. CD19 was sorted from human PBMC (Hemacare, PB 009C-3) using a CD19 immunomagnetic bead kit (Miltenyi Biotec, 130-050-301) according to the kit instructions ⁺ B cells.

B cells obtained by the above-described sorting were cultured in RPMI1640 medium containing 10% fetal bovine serum (Lifetechnologies, 10091148), and 10ng/mL of IL4 was added. B cells were grown at 1X 10 ⁵ Number of individuals/wells was seeded in 96-well plates and NK003 at different concentrations was added: 0.01. Mu.g/ml, 0.05. Mu.g/ml, 0.1. Mu.g/ml, 0.5. Mu.g/ml, 1. Mu.g/ml, 10. Mu.g/ml and 100. Mu.g/ml HEL as negative control. After 48h of co-incubation at 37 ℃, proliferation of cells was detected using CellTiter-Glo kit (Promega, G7570): mu.l of cells were taken and 100. Mu.l added according to the kit instructions Reagent, mixing, standing for 10min under dark condition, and using enzyme labeling instrumentThe higher the intensity of the read fluorescence, the stronger the proliferation of the cells. Curves were fitted using GraphPad Prism 6.0 and EC50 was calculated. As a result, as shown in FIG. 11, NK003 can promote proliferation of human B cells with an EC50 of 4nM.

6.3 B cell activation

Human Peripheral Blood Mononuclear Cells (PBMC) were used to examine the ability of NK003 to activate B cells.

Sorting CD19 from human PBMC with CD19 immunomagnetic bead kit as shown in 6.2 ⁺ B cells. The B cells obtained by the above-described sorting were cultured in RPMI 1640 medium containing 10% fetal bovine serum (Lifetechnologies, 10091148). B cells were grown at 1X 10 ⁵ Number of individuals/wells was seeded in 96-well plates and NK003 at different concentrations was added: as negative controls, HEL was used at 0.01. Mu.g/ml, 0.05. Mu.g/ml, 0.1. Mu.g/ml, 0.5. Mu.g/ml, 1. Mu.g/ml, 10. Mu.g/ml and 100. Mu.g/ml. For crosslinking, the above groups were further added with goat anti-human Fc at a concentration of 2.5. Mu.g/ml. After 48h of incubation, PBS was washed three times, PE-CD86 (B cell activation marker molecule, biolegend, 105007) was added 1:100 and incubated for 30min at 4℃in the absence of light. B cell activation after antibody addition (both cross-linked and non-cross-linked) was measured using a flow cytometer and expressed as MFI for APC-CD 86. Curves were fitted using GraphPad Prism 6.0 and EC50 was calculated. As shown in fig. 12A and B, NK003 activated human B cells with an EC50 of 70Nm (fig. 12A) without the addition of a cross-linking agent (goat anti-human Fc, anti-Fc), with an EC50 of 4Nm (fig. 12B) with the addition of a cross-linking agent, and the intensity of the cross-linked form of NK003 activated B cells was significantly increased (CD 86 expression significantly higher) than that without cross-linking.

Example 7 in vivo characterization of human CD40 antibody NK003

Different in vivo experiments were performed to further characterize the effect of NK003 human antibodies.

7.1 NK003 inhibits tumor growth in SCID mice

Antibody-dependent cell-mediated cytotoxicity (ADCC) refers to the binding of the Fab fragment of an antibody to an epitope of a tumor cell, the binding of its Fc fragment to an FcR on the surface of a killer cell (NK cell, macrophage, etc.), which mediates the direct killing of the target cell by the killer cell. To evaluate NK 003-mediated ADCC killing of tumor cells, we used SCID mice to vaccinate tumor cells expressing CD 40.

Eight male SCID mice five weeks old were purchased from velocin and housed in SPF-rated animal houses. After one week of breeding, the breeding is divided into two groups randomly, and the back is 2.5X10 ⁶ The number of individuals/individuals was inoculated subcutaneously with Raii cells, four for each group, and the Day 0. The first group was dosed with NK003 antibody and the second group was dosed with HEL control antibody.

From Day 1, NK003 was intraperitoneally injected at a dose of 7.5mg/kg, administered once every 7 days three times, HEL as a control antibody, and the dose and number of administrations were the same as NK003. The mice were weighed every three days and the vertical size of the tumor was measured with a vernier caliper, the volume of the tumor = (length x width)/2, and a tumor growth curve was drawn.

The results showed that no significant tumor was seen in the NK003 group mice compared to the control group (fig. 13A), and that the survival rate of the NK003 group mice was 100% (fig. 13B). NK003 can remarkably inhibit the growth of tumors by mediating ADCC action, and improve the survival rate of mice.

7.2 NK003 activation of the immune system in CD40 humanized mice

In the C57BL/6 genetic background, human CD40 and Fc receptors FcgammaRIIA, fcgammaRIIB and FcgammaRIIIA are inserted into the genome of a mouse, and the expression of the human CD40 and Fc receptor proteins is simultaneously replaced by the expression of the endogenous CD40 and Fc receptor of the mouse. CD40 humanized mouse model was constructed and provided for Shanghai university of traffic Li Fubin and was bred in SPF class animal houses. OT-1 mice were supplied from Shanghai university of traffic Li Fubin and kept in SPF-class animal houses. OT-1 mice were sacrificed and spleens were removed from cervical dislocation, and crushed spleens were squeezed to collect spleen cells. Kit using immunomagnetic beads (R)&D Systems, MAGM 203), CD8 was isolated according to the kit instructions ⁺ T cells, i.e. OT-1 CD8 ⁺ T cells.

Eight week old male CD40 humanized mice were selected at 2X 10 ⁶ Quantity of individual/individual tail intravenous injections of OT-1 CD8 ⁺ T cells were simultaneously intraperitoneally injected with 0.1mg/kg DEC-OVA (Sigma-Aldrich, SAB 4700735) and 5mg/kg NK003 (HEL at the same dose as control antibody). Seven days after cell inoculation, mice were sacrificed by cervical vertebrae to isolate spleens. Grinding spleen to obtain single cell suspension, and removing by lysis method After erythrocytes, OVA-specific OT-1CD8 was detected by flow cytometry using anti-CD 4 (ebioscience, RM 4-5), anti-CD 8 (Biolegend, 53-6.7), anti-CD 45.1 (Biolegend, A20) and anti-TCR-V.alpha.2 (Biolegend, B20.1) staining ⁺ Proliferation of T cells. CD45.1 ⁺ CD8 ⁺ TCR-Vα2 ⁺ The subgroup is OT-1CD8 ⁺ T cells (OT 1 cells). CD45.1 ⁺ CD8 ⁺ The subset was CD8 cells, CD45.1 ⁺ CD4 ⁺ The subpopulation is CD4 cells. The results are shown in FIG. 14, group NK003 OT-1CD8 ⁺ The T cell fraction increased significantly and the cell number increased significantly, as well as the CD8 to CD4 ratio. NK003 can activate the CD40 humanized mouse immune system.

7.3 NK003 inhibits tumor growth in CD40 humanized mice

To evaluate the tumor-inhibiting effect of NK003 activating immune system, we used CD40 humanized mice constructed and provided at Shanghai university of traffic Li Fubin as described in 7.2 for inoculation of tumor cells MC38 (basic medical cell center of basic medical institute of the national academy of medicine, 3111C0001CCC 000523).

The positive control anti-CD 40 antibody CP870893 sequences used herein are from patent US 7338660 (see SEQ ID NO:46 and SEQ ID NO:48 for light and heavy chain sequences). The Jin Weizhi synthesized CP870893 light and heavy chains were transfected into pFUSE vectors and 293F suspension cells were transiently co-transfected 1:1 to express full length antibodies and after 1 week expression were used on the AKTA system using Superdex ^TM 200 The purification of the incoase pre-packed column (specific procedure is described in example 4).

The backs of eight-week-old male CD40 humanized mice were treated to 2X 10 ⁶ The MC38 cells were inoculated subcutaneously in the number of individuals/individual, when tumors grew to about 100mm ³ The randomization was divided into three groups: the HEL control group was composed of 8 animals, five animals each, NK003 and CP870893 (positive control), and the antibody was administered by intraperitoneal injection at a dose of 3mg/kg, once every 3 days, twice. The mice were weighed every three days and the vertical size of the tumor was measured with a vernier caliper, the volume of the tumor = (length x width)/2, and the tumor growth curve and the mice weight curve were plotted. Tumor inhibition rate = (control mean volume-experimental mean volume)/control mean volume x 100%.

The results are shown in fig. 15A, where NK003 tumor inhibition was 75% at day 18, whereas CP870893 tumor inhibition was only 60%. At the same time, we examined the body weight of mice, as shown in fig. 15B, without significant decrease in the body weight of mice using NK003 antibody. Thus, NK003 can significantly inhibit tumor growth and has no significant effect on mouse body weight.

Example 8 increased binding to FcgammaRIIB and CD40 agonist Activity with the NK003-V12, NK003-S267E/L328F Fc region mutant

To increase binding to fcγr and increase agonist activity of NK003 antibodies, residue glutamic acid (E) at residue 233 of the NK003 Fc region (IgG 1, EU numbering) was mutated to aspartic acid (D), residue glycine (G) at residue 237 was mutated to aspartic acid (D), residue histidine (H) at residue 268 was mutated to aspartic acid (D), residue proline (P) at residue 271 was mutated to glycine (G), residue alanine (a) at residue 330 was mutated to arginine (R), resulting in NK003-V12 mutants. The 267 th residue serine (S) of NK003 Fc region (IgG 1, EU numbering) was mutated to glutamic acid (E), and the 328 th residue leucine (L) was mutated to phenylalanine (F), resulting in NK003-S267E/L328F mutant (sequence see sequence table). The NK003-V12 and NK003-S267E/L328F mutants were confirmed by sequencing after construction.

TABLE 3 summarisation of mutant mutation sites of the NK003 Fc region

To evaluate agonist activity of NK003-V12 and NK003-S267E/L328F mutants, light and heavy chains of the NK003-V12 and NK003-S267E/L328F mutants synthesized by Jin Weizhi were transfected into pFUSE vectors, 293F suspension cells were transiently co-transfected 1:1, full length antibodies were expressed, and Superdex was used on the AKTA system after 1 week of expression ^TM 200 IncreatePre-packed column purification (specific procedure is described in example 4).

The Jin Weizhi synthetic fcγriia (NCBI, nm_ 001136219.1) and fcγriib (NCBI, nm_ 004001.4) CDS regions were cloned into pCDH vectors, transiently transfected into 293FT cells with PEI using standard procedures, and after 6h of transfection, the cells were incubated in DMEM medium containing 10% fetal bovine serum (Biological Industries, 04-001-1A) at 37 ℃ to obtain fcγriia-expressing cells and fcγriib-expressing 293FT cells, respectively, after 48h of expression.

Respectively take 2X 10 as above ⁵ Each tube 293 FT-FcgammaRIIA or 293 FT-FcgammaRIIB cells was added 2X 10 each ⁵ Individual/tube NF-. Kappa.B-GFP+hCD 40 reporter cells (described in example 1). Different concentrations of NK003, NK003-V12 and NK003-S267E/L328F diluted in PBS were added to each tube: 0.01. Mu.g/ml, 0.05. Mu.g/ml, 0.1. Mu.g/ml, 0.5. Mu.g/ml, 1. Mu.g/ml, 5. Mu.g/ml and 10. Mu.g/ml HEL as negative controls; after 24h co-incubation at 37℃PBS was washed three times and analyzed by flow cytometry to detect the MFI of GFP. As a result, as shown in FIG. 16, the agonist activity of NK003-V12, NK003-S267E/L328F was significantly enhanced as compared with that of wild type NK 003.

Example 9 affinity maturation of human CD40 antibody NK003

Mutant libraries were constructed for the complementarity determining regions and framework regions of NK003VH, VL, respectively, and were inserted into pcomb3 phage vector (Biovector Inc. 108925) in Fab format (FIG. 17), and optimization of NK003 was accomplished using phage affinity maturation methods.

9.1 NK003VH, VL complementarity determining region CDR3 affinity maturation

9.1.1 Construction of NK003VH and VL complementarity determining region CDR3 mutant library

NK003 light chain DNA (SEQ ID NO: 72) with a CDR3 inserted into a stop codon was used as a template, and light chain VL and CL fragments were amplified using primer pairs VL1/VL2 and CL1/CL2, respectively. Reaction conditions: 2min at 95 ℃ and 1cycle;95℃30s,65℃30s,72℃10s,35 cycles; 72℃for 5min,1cycle. The PCR target fragment was recovered using the Tiangen recovery kit. The primers were as follows:

VL1：5’-GCGGCCGAGCTCGATGTTGTGATGACTCAG-3’

VL2：5’-GGTCCCCTGGCCAAA AGT GTA CGG AGT TCA TAG ACC TTG CATGCAGTAATAAAG-3' (MNN was mutated at two arbitrary positions at the transversal line, synthesized by Jin Weizhi company in high throughput)

CL1：5’-TTTGGCCAGGGGACCAAGCTGGAGATC-3’

CL2：5’-TATCTAGATTAATTAAATCACTCTCCCCTGTTGAAGCTC-3’

And performing overlap PCR amplification on the two parts of PCR products to obtain an NK003 light chain mutation library. Reaction conditions: 2min at 95 ℃ and 1cycle;95 ℃ for 30s,65 ℃ for 30s,72 ℃ for 20s,8 cycles; adding VL1/CL2 primer, 95 ℃ for 30s,65 ℃ for 30s,72 ℃ for 20s and 27cycle;72℃for 5min,1cycle. Double digestion of NK003 light chain mutant library and pcomb3 vector with SacI (NEB, R3156L) and PacI (NEB, R0547L) was performed, respectively, and the PCR fragment of interest was recovered using a Tiangen recovery kit. The mutant library genes and vector were ligated at 25℃for 3h via T4 ligase (NEB, M0202L) at a molar ratio of 3:1. The ligation products were electrotransformed into XL1-Blue electrotransformation competence using electrotransformation to construct a pool size of 5X 10 ⁵ The light chain mutant library was named pcomb3-NK003-LCDR3.

NK003 heavy chain DNA (SEQ ID NO: 73) with a CDR3 inserted into a stop codon was used as a template, and heavy chain VH and CH1 fragments were amplified using primer pairs VH1/VH2 and CH1-1/CH1-2, respectively. Reaction conditions: 2min at 95 ℃ and 1cycle;95℃30s,65℃30s,72℃10s,35 cycles; 72℃for 5min,1cycle. The primers were as follows:

VH1：5’-TGCAGCTGCTCGAGCAGGTACAGCTGGTGCAGTC-3’

VH2：5’-CTTTGCCCCAGACGTC CAT GTA GTA GTA GTA GGT TCA AGT AGC TCC CAC TCT TTCTCTCGCACAG-3' (MNN was mutated at two arbitrary positions at the transversal line, synthesized by Jin Weizhi company in high throughput)

CH1-1：5’-GACGTCTGGGGCAAAGGGACCACGGTC-3’

CH1-2：5’-GCCTGGCCACTAGTTTTGTCAACTTTCTTGTCC-3’

And (3) performing overlap PCR amplification on the two parts of PCR products to obtain an NK003 heavy chain mutant library, wherein the reaction conditions are as follows: 2min at 95 ℃ and 1cycle;95 ℃ for 30s,65 ℃ for 30s,72 ℃ for 20s,8 cycles; adding VH1/CH1-2 primer, 95 ℃ 30s,65 ℃ 30s,72 ℃ 20s,27cycle;72℃for 5min,1cycle. The NK003 heavy chain mutant library and pcomb3-NK003-LCDR3 vector were digested with SpeI (NEB, R3133L) and XhoI (NEB, R0146L), respectively, and the PCR fragment of interest was recovered using a root recovery kit. The mutant library genes and the vector are connected for 3 hours at 25 ℃ through T4 ligase according to the mol ratio of 3:1. Construction of ligation products into XL1-Blue electrotransformation competence using electrotransformationThe stock capacity was 2.4X10 ⁷ The light and heavy chain double mutant library is named pcomb3-NK003-AM.

9.1.2 phage mutant antibody library screening

Antibodies binding to CD40 were screened using the pcomb3-NK003-AM antibody library, for specific screening methods, see example 2. The screening results are shown in Table 1.

TABLE 12 screening of CD40 binding antibodies from pcomb3-NK003-AM antibody libraries using phage display technology

To initially evaluate the positive rate of screening for CD40 binding antibodies, we picked the third round of 32 phage monoclonal for phage ELISA analysis. Specific phage ELISA methods are described in example 2. More than 3-fold higher CD40 binding signal than BSA control was defined as positive clones. The results are shown in FIG. 18, with a third round of screening positivity of 96.8%.

The phage eluted in the third round is infected with XL1-blue and then coated, and after being scraped evenly, NK003-AM plasmid is extracted by using a root plasmid small extraction kit. After double cleavage with SacI and SpeI, the target fragment of about 1500bp was recovered and sent to Jin Weizhi for three-generation sequencing. The sequencing results are shown in FIG. 19. According to the third generation sequencing result, the screened antibodies NK003-AM-9, NK003-AM-18 heavy chain and light chain DNA (Jin Weizhi) are synthesized and cloned into a vector pFUSE respectively, and the specific method for purifying expression is shown in example 4.NK003-AM-9 and NK003-AM-18VH/VL sequences are shown in the sequence tables 6 to 8.

9.1.3 NK003 CDR3 affinity maturation activity assay

In this example, NK003-AM-9 and NK003-AM-18 antibodies activate NF- κB-GFP+hCD40 reporter cell lines by flow cytometry, and the specific steps are as follows:

respectively taking 3×10 ⁵ The individual/tubes were fine reported as obtained in example 1 for NF-. Kappa.B-GFP+hCD 40Cells, NK003-AM-9, NK003-AM-18 and NK003 were added at different concentrations, respectively: 0.01. Mu.g/ml, 0.05. Mu.g/ml, 0.1. Mu.g/ml, 0.5. Mu.g/ml and 1. Mu.g/ml; for cross-linking, the secondary antibodies, goat anti-human Fc (SouthernBiotech, SBA-2048-01), were added simultaneously at a concentration of 2.5. Mu.g/ml for each group. After co-incubation in RPMI 1640 medium (Lifetechnologies, C11875500 CP) containing 10% fetal bovine serum (Biological Industries, 04-001-1A) at 37℃for 24h, PBS was washed three times and analyzed using a flow cytometer. The obtained data were fitted to a curve using GraphPad Prism 6.0 and EC50 was calculated. As a result, as shown in FIG. 20, the NK003-AM-9 and NK003-AM-18 activated NF-. Kappa.B-GFP+hCD 40 reporter cell lines in a cross-linked form with EC50 of 0.25nM and 0.3nM, respectively, and the activities were similar and increased by nearly 10-fold over wild-type NK 003.

9.2 NK003 VH and VL framework affinity maturation

9.2.1 Construction of NK003 VH and VL framework region mutation library

Nest PCR was performed on the light chain VL region using NK003 light chain DNA (SEQ ID NO: 74) as a template, using a random mutagenesis PCR kit (Agilent Technologies, 200550) and primer pairs VL1/VL2, VL3/VL4, reaction conditions: 2min at 95 ℃ and 1cycle;95 ℃ 30s,65 ℃ 30s,72 ℃ 30s,28cycle;72℃for 10min,1cycle. The NK003 light chain DNA (SEQ ID NO: 11) was used as a template to amplify the light chain CL region using the primer pair CL1/CL2, and the reaction conditions were: 2min at 95 ℃ and 1cycle;95 ℃ for 30s,65 ℃ for 30s,72 ℃ for 30s,30cycle;72℃for 5min,1cycle. The PCR target fragment was recovered using the Tiangen recovery kit. The primers were as follows:

VL1：5’-CTATCGCGATTGCAGTGGCACTGGCTG-3’

VL2：5’-CCAGATTTCAACTGCTCATCAGATGGC-3’

VL3：5’-CTACCGTGGCCCAGGCGGCCGAGCTC-3’

VL4：5’-GAAGACAGATGGTGCAGCCACAGTTCG-3’

CL1：5’-CGAACTGTGGCTGCACCATCTGTCTTC-3’

CL2：5’-TATCTAGATTAATTAAATCACTCTCCCCTGTTGAAGCTC-3’

And performing overlap PCR amplification on the yL and CL PCR products to obtain an NK003 light chain mutation library. Reaction conditions: 2min at 95 ℃ and 1cycle;95 ℃ for 30s,65 ℃ for 30s,72 ℃ for 40s,8 cycles; adding VL3/CL2 primer, 95 ℃ for 30s,65 ℃ for 30s,72 ℃ for 40s and 27cycle;72℃for 10min,1cycle. Double digestion of NK003 light chain mutant library and pcomb3 vector was performed with SacI and PacI, respectively, and PCR target fragment was recovered using a Tiangen recovery kit. The mutant library genes and the vector are connected for 3 hours at 25 ℃ through T4 ligase according to the mol ratio of 3:1. The ligation products were electrotransformed into XL1-Blue electrotransformation competence using electrotransformation to construct a library capacity of 1X 10 ⁵ The light chain mutant library was named pcomb3-NK003-VL.

Nested PCR amplification of heavy chain VH region was performed using NK003 heavy chain DNA (SEQ ID NO: 75) as template and primer pairs VH1/VH2, VH3/VH4, reaction condition 3: 2min at 95 ℃ and 1cycle;95 ℃ 30s,65 ℃ 30s,72 ℃ 30s,28cycle;72℃for 10min,1cycle. The NK003 heavy chain DNA (SEQ ID NO: 12) was used as a template, and the heavy chain CH1 region was amplified using the primer pair CH1-1/CH1-2, and the reaction conditions were: 2min at 95 ℃ and 1cycle;95 ℃ 30s,65 ℃ 30s,72 ℃ 30s,32 cycles; 72℃for 10min,1cycle. The PCR target fragment was recovered using the Tiangen recovery kit. The primers were as follows:

VH1：5’-GCCGCTGGATTGTTATTACTCGCTGC-3’

VH2：5’-CAGAGGTGCTCTTGGAGGAGGGTGCC-3’

VH3：5’-GCCATGGCCGAGGTGCAGCTGCTCGAG-3’

VH4：5’-GAAGACCGATGGGCCCTTGGTGGAGGC-3’

CH1-1：5’-GCCTCCACCAAGGGCCCATCGGTCTTC-3’

CH1-2：5’-GCCTGGCCACTAGTTTTGTCAACTTTCTTGTCC-3’

And performing overlap PCR amplification on the VH and CH1 PCR products to obtain an NK003 heavy chain mutation library. Reaction conditions: 2min at 95 ℃ and 1cycle;95 ℃ 30s,65 ℃ 30s,72 ℃ 40s,8 cycles; adding VH3/CH1-2 primer, 95 ℃ 30s,65 ℃ 30s,72 ℃ 40s,27cycle;72℃for 10min,1cycle. SpeI and XhoI were used to double-cleave NK003 heavy chain mutant library and pcomb3-NK003-VL vector, respectively, and the PCR target fragment was recovered using a Tiangen recovery kit. The mutant library genes and the vector are connected for 3 hours at 25 ℃ through T4 ligase according to the mol ratio of 3:1. The ligation products were electrotransformed into XL1-Blue electrotransformation competence using electrotransformation to construct a library capacity of 2.4X10 ⁷ The light and heavy chain double mutant library is named pcomb3-NK003-EP.

9.2.2 phage mutant antibody library screening

Antibodies binding to CD40 were screened using the pcomb3-NK003-EP antibody library, for specific screening methods see example 2. The screening results are shown in Table 2.

TABLE 13 screening of CD40 binding antibodies from pcomb3-NK003-EP antibody library Using phage display technology

The phage eluted in the third round is infected with XL1-blue and then coated, and after being scraped evenly, NK003-EP plasmid is extracted by using a root plasmid small extraction kit. After double cleavage with SacI and SpeI, the target fragment of about 1500bp was recovered and sent to Jin Weizhi for three-generation sequencing. The sequencing results are shown in FIG. 21. According to the third generation sequencing result, the antibody NK003-AM-18-EP1 heavy chain DNA (Jin Weizhi) was synthesized and cloned into the vector pFUSE. Plasmids containing NK003-AM-18 light chain and NK003-AM-18-EP1 heavy chain, respectively, were transiently co-transfected into 293F suspension cells at 1:1 using standard procedures with PEI to express full length NK003-AM-18-EP1 antibody. The specific method of purification expression is described in example 4.NK003-AM-18-EP1 VH sequence is shown in a sequence table 6.

9.2.3 Identification of NK003 framework region affinity maturation Activity

In this example, NK003-AM-18-EP1 antibody activated NF-. Kappa.B-GFP+hCD 40 reporter cell line was examined by flow cytometry as follows:

respectively taking 3×10 ⁵ NF-. Kappa.B-GFP+hCD 40 reporter cells were obtained as in example 1 and were added to different concentrations of NK003-AM-18, NK003-AM-18-EP1 and NK003:0.0075nM, 0.025nM, 0.075nM, 0.25nM, 0.75nM, 2.5nM and 7.5nM; for cross-linking, the secondary antibodies, goat anti-human Fc, were added simultaneously at a concentration of 2.5. Mu.g/ml for each group. After co-incubation in RPMI 1640 medium (Lifetechnologies, C11875500 CP) containing 10% fetal bovine serum (Biological Industries, 04-001-1A) at 37℃for 24h, PBS was washed three times and analyzed using a flow cytometer. The data obtained uses GraphPad Prism 6.0 fits the curve and calculates the EC50. Results As shown in FIG. 22, NK003-AM-18-EP1 activated the NF-. Kappa.B-GFP+hCD 40 reporter cell line in cross-linked form with an EC50 of 0.35nM, similar to NK003-AM-18 activity.

Claims

1. An antibody or antigen-binding fragment thereof that binds CD40 comprising

(i) HCDR1, HCDR2 and HCDR3 consisting of the following sequences: SEQ ID NO. 1, SEQ ID NO. 3 and SEQ ID NO. 5, LCDR1, LCDR2 and LCDR3 consisting of the sequences: SEQ ID NO. 7, SEQ ID NO. 9 and SEQ ID NO. 11; or alternatively

(ii) HCDR1, HCDR2 and HCDR3 consisting of the following sequences: SEQ ID NO. 1, SEQ ID NO. 3 and SEQ ID NO. 51, LCDR1, LCDR2 and LCDR3 consisting of the sequences: SEQ ID NO. 7, SEQ ID NO. 9 and SEQ ID NO. 53; or alternatively

(iii) HCDR1, HCDR2 and HCDR3 consisting of the following sequences: SEQ ID NO. 1, SEQ ID NO. 3 and SEQ ID NO. 52, LCDR1, LCDR2 and LCDR3 consisting of the sequences: SEQ ID NO. 7, SEQ ID NO. 9 and SEQ ID NO. 54.

2. The antibody or antigen-binding fragment thereof of claim 1, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein

(i) The heavy chain variable region VH consists of the amino acid sequence of SEQ ID NO. 13, and the light chain variable region VL consists of the amino acid sequence of SEQ ID NO. 15;

(ii) The heavy chain variable region VH consists of the amino acid sequence of SEQ ID NO. 58 and the light chain variable region VL consists of the amino acid sequence of SEQ ID NO. 64;

(iii) The heavy chain variable region VH consists of the amino acid sequence of SEQ ID NO. 60, and the light chain variable region VL consists of the amino acid sequence of SEQ ID NO. 66; or (b)

(iv) The heavy chain variable region VH consists of the amino acid sequence of SEQ ID NO. 62 and the light chain variable region VL consists of the amino acid sequence of SEQ ID NO. 66.

3. The antibody or antigen-binding fragment thereof of claim 1, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain and a light chain, wherein

(i) The heavy chain consists of the amino acid sequence of SEQ ID NO. 17, and the light chain consists of the amino acid sequence of SEQ ID NO. 21;

(ii) The heavy chain consists of the amino acid sequence of SEQ ID NO. 18, and the light chain consists of the amino acid sequence of SEQ ID NO. 21;

(iii) The heavy chain consists of the amino acid sequence of SEQ ID NO. 19, and the light chain consists of the amino acid sequence of SEQ ID NO. 21;

(iv) The heavy chain consists of the amino acid sequence of SEQ ID NO. 67, and the light chain consists of the amino acid sequence of SEQ ID NO. 68;

(v) The heavy chain consists of the amino acid sequence of SEQ ID NO. 69 and the light chain consists of the amino acid sequence of SEQ ID NO. 71; or (b)

(vi) The heavy chain consists of the amino acid sequence of SEQ ID NO. 70 and the light chain consists of the amino acid sequence of SEQ ID NO. 71.

4. The antibody or antigen-binding fragment thereof of claim 2, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain and a light chain, wherein

5. The antibody or antigen-binding fragment thereof of any one of claims 1 to 4, wherein the antibody is a monoclonal antibody.

6. The antibody or antigen-binding fragment thereof of any one of claims 1 to 4, wherein the antibody is a humanized antibody or a human antibody or a chimeric antibody.

7. The antibody or antigen-binding fragment thereof of any one of claims 1 to 4, wherein the antigen-binding fragment is an antibody fragment selected from the group consisting of: fab, fab '-SH, fv, single chain antibody, (Fab') ₂ A single domain antibody, diabody (dAb), or a linear antibody.

8. The antibody or antigen-binding fragment thereof of claim 7, wherein the single chain antibody is an scFv.

9. An isolated nucleic acid encoding any one or more chains of the antibody or antigen-binding fragment thereof of any one of claims 1 to 8.

10. A vector comprising the nucleic acid of claim 9.

11. The vector of claim 10, wherein the vector is an expression vector.

12. A host cell comprising the nucleic acid of claim 9 or the vector of claim 10 or 11.

13. The host cell of claim 12, wherein the host cell is prokaryotic or eukaryotic.

14. The host cell of claim 12, wherein the host cell is selected from the group consisting of a yeast cell, a mammalian cell, or other cell suitable for the production of antibodies or antigen-binding fragments thereof.

15. The host cell of claim 12, wherein the host cell is a CHO cell or 293 cell.

16. The host cell of claim 15, wherein the CHO cell is a CHOs cell, a CHOK1SV cell or a CHOK1SV GS-KO cell.

17. The host cell of claim 15, wherein the 293 cell is a HEK293 cell.

18. A method of making an antibody or antigen-binding fragment thereof that binds CD40, the method comprising culturing a host cell comprising a nucleic acid encoding the antibody or antigen-binding fragment thereof of any one of claims 1-8 under conditions suitable for expression of the antibody.

19. The method of claim 18, wherein the method further comprises recovering the antibody or antigen-binding fragment thereof from the host cell.

20. An immunoconjugate comprising the antibody or antigen-binding fragment thereof of any one of claims 1 to 8 and other substances.

21. The immunoconjugate of claim 20, wherein said other substance is a therapeutic agent or label.

22. The immunoconjugate of claim 21, wherein said therapeutic agent is selected from the group consisting of a chemotherapeutic agent, other antibodies, cytotoxic agent, vaccine, anti-infective active agent, small molecule drug, or immunomodulator

23. A pharmaceutical composition comprising an antibody or antigen-binding fragment thereof according to any one of claims 1 to 8 or an immunoconjugate according to any one of claims 20 to 22, and optionally a pharmaceutical excipient.

24. The pharmaceutical composition of claim 23, further comprising one or more additional therapeutic agents.

25. The pharmaceutical composition of claim 24, wherein the therapeutic agent is selected from the group consisting of a chemotherapeutic agent, other antibodies, a cytotoxic agent, a vaccine, an anti-infective agent, a small molecule drug, or an immunomodulator.

26. A combination product comprising an antibody or antigen-binding fragment thereof according to any one of claims 1 to 8 or an immunoconjugate according to any one of claims 20 to 22, and one or more other therapeutic agents.

27. The combination product of claim 26, wherein the therapeutic agent is selected from the group consisting of a chemotherapeutic agent, other antibodies, a cytotoxic agent, a vaccine, an anti-infective agent, a small molecule drug, or an immunomodulator.

28. Use of the antibody or antigen-binding fragment thereof of any one of claims 1-8, or the immunoconjugate of any one of claims 20 to 22, or the pharmaceutical composition of any one of claims 23 to 25, or the combination product of claim 26 or 27, for the manufacture of a medicament for preventing or treating a CD 40-associated disease in a subject, wherein the CD 40-associated disease is a CD 40-associated tumor.

29. The use of claim 28, wherein the tumor is selected from the group consisting of lymphoma, colon cancer, colorectal cancer, rectal cancer, lung cancer, liver cancer, gastric cancer, and metastatic cancers thereof.

30. The use of claim 29, wherein the lung cancer is non-small cell lung cancer.

31. The use of any one of claims 28-30, wherein the medicament is administered to the subject in combination with one or more therapies.

32. The use of claim 31, wherein the therapy is a therapeutic regimen and/or other therapeutic agent.

33. The use of claim 32, wherein the treatment modality comprises surgical treatment and/or radiation therapy.

34. The use of claim 32 or 33, wherein the additional therapeutic agent is selected from the group consisting of a chemotherapeutic agent, an additional antibody, a cytotoxic agent, a vaccine, an anti-infective agent, a small molecule drug, or an immunomodulator.

35. A test kit comprising the antibody or antigen-binding fragment thereof of any one of claims 1-8 or the immunoconjugate of any one of claims 20 to 22.